Recombinant Antibody Composition

ABSTRACT

The present invention relates to a recombinant antibody composition having higher complement-dependent cytotoxic activity than a human IgG1 antibody and a human IgG3 antibody, wherein a polypeptide comprising a CH2 domain in the Fc region of a human IgG1 antibody is replaced by a polypeptide comprising an amino acid sequence which corresponds to the same position of a human IgG3 antibody indicated by the EU index as in Kabat, et al.; a DNA encoding the antibody molecule or a heavy chain constant region of the antibody molecule contained in the recombinant antibody composition; a transformant obtainable by introducing the recombinant vector into a host cell; a process for producing the recombinant antibody composition using the transformant; and a medicament comprising the recombinant antibody composition as an active ingredient.

CROSS REFERENCES

This is a continuation application of U.S. patent application Ser. No.11/491,501, filed Jul. 24, 2006, which is based on U.S. ProvisionalPatent Applications No. 60/701,477 filed Jul. 22, 2005 and 60/791,213filed Apr. 12, 2006, and Japanese Patent Applications No. 2005-212979filed Jul. 22, 2005, and 2006-108216 filed Apr. 11, 2006, the entirecontents of which are incorporated hereinto by reference. All referencescited herein are incorporated in their entirety.

FIELD OF THE INVENTION

The present invention relates to a recombinant antibody compositionhaving higher complement-dependent cytotoxic activity than a human IgG1antibody and a human IgG3 antibody, wherein a polypeptide comprising aCH2 domain in the Fc region of a human IgG1 antibody is replaced by apolypeptide comprising an amino acid sequence which corresponds to thesame position of a human IgG3 antibody indicated by the EU index as inKabat, et al. (hereinafter referred to as EU index); a DNA encoding theantibody molecule or a heavy chain constant region of the antibodymolecule contained in the recombinant antibody composition; atransformant obtainable by introducing the DNA into a host cell; aprocess for producing the recombinant antibody composition using thetransformant; and a medicament comprising the recombinant antibodycomposition as an active ingredient.

BACKGROUND OF THE INVENTION

Since antibodies have high binding activity, binding specificity andhigh stability in blood, applications thereof to diagnostic, preventiveand therapeutic agents for various human diseases have been attempted(Non-patent Reference 1). In addition, human chimeric antibodies orhumanized antibodies have been prepared from non-human animal antibodiesusing gene recombination techniques (Non-patent References 2 to 5). Thehuman chimeric antibody is an antibody in which its variable region isan antibody of non-human animal and its constant region is a humanantibody. The humanized antibody is an antibody in which thecomplementarity determining region (hereinafter referred to as CDR) of anon-human animal is replaced by CDR of a human antibody.

The human chimeric antibodies and humanized antibodies have resolvedproblems possessed by mouse antibodies and the like, such as the highimmunogenicity, low effector function and short blood half-life ofnon-human animal antibodies, and applications of monoclonal antibodiesto pharmaceutical preparations were made possible by using them(Non-patent References 6 to 9). In the Unites States, for example,plurality of humanized antibodies have already been approved as anantibody for cancer treatment, and on the market (Non-patent Reference10).

These human chimeric antibodies and humanized antibodies actually showeffects to a certain degree at clinical level, but therapeuticantibodies having higher effects are in demand. For example, in the caseof single administration of Rituxan™ (Non-patent Reference 11)(manufactured by IDEC/Roche/Genentech) which is a human chimericantibody to CD20, it has been reported that its response ratio forrecurrent low malignancy non-Hodgkin lymphoma patients by the phase IIIclinical test is no more than 48% (complete remission 6%, partialremission 42%), and its average duration of response is 12 months(Non-patent Reference 12). In the case of combination use of Rituxan™and chemotherapy (CHOP: Cyclophosphamide, Doxorubicin, Vincristine), ithas been reported that its response ratio for recurrent low malignancyand follicular non-Hodgkin lymphoma patients by the phase II clinicaltest is 95% (complete remission 55%, partial remission 45%), but sideeffects due to CHOP were found (Non-patent Reference 13). In the case ofsingle administration of Herceptin™ (manufactured by Genentech) which isa humanized antibody to HER2, it has been reported that its responseratio for metastatic breast cancer patients by the phase III clinicaltest is only 15%, and its average duration of response is 9.1 months(Non-patent Reference 14).

The human antibody molecule is also called immunoglobulin (hereinafterreferred to as Ig) and classified into respective classes of IgA, IgD,IgE, IgG and IgM based on its molecular structure. The antibody moleculeof human IgG (hereinafter referred to as IgG) mainly used as thetherapeutic antibody is formed by two respective polypeptides calledheavy chain (hereinafter referred to as H chain) and light chain(hereinafter referred to as L chain). The H chain is formed byrespective domain structures called H chain variable region (hereinafterreferred to as VH), CH1, hinge, CH2 and CH3, from the N-terminal side.The respective domains CH1, hinge, CH2 and CH3 are also called heavychain constant region as a whole (hereinafter referred to as CH), andthe CH2 and CH3 domains are also called Fc region as a whole. The Lchain is formed by respective domain structures called L chain variableregion (hereinafter referred to as VL) and L chain constant region(hereinafter referred to as CL), from the N-terminal side.

Four subclasses including IgG1, IgG2, IgG3 and IgG4 exist in the IgGantibody H chain. The H chains of respective IgG subclasses mutuallyhave about 95% homology of amino acid sequence in the constant regionexcluding the hinge which is rich in variability (FIG. 1).

Regardless of the high homology of amino acid sequences in respectiveIgG subclasses, height of the biological activity possessed therebyvaries (Non-patent Reference 15). The biological activity includeseffector functions such as complement-dependent cytotoxic activity(hereinafter referred to as CDC), antibody-dependent cell-mediatedcytotoxic activity (hereinafter referred to as ADCC) and phagocytoticactivity, and these functions play an important role in the living body,such as exclusion of foreign matters and pathogens.

A family of Fcγ receptor (hereinafter referred to as FcγR) are expressedon the surface of various leukocytes such as natural killer cell(hereinafter referred to as NK cell), monocyte, macrophage andgranulocyte. The FcγR is classified into active type FcγR includingFcγRI, FcγRIIa, FcγRIIIa and FcγRIIIb and suppression type FcγR ofFcγRIIb. IgG antibodies, particularly IgG1 and IgG3 in human, stronglybind to these receptors and induce ADCC activity and phagocytoticactivity by leukocytes as a result.

The ADCC activity is a cytolytic reaction in which an antibody bound toits antigen binds to mainly FcγRIIIa on the NK cell surface via Fcmoiety, and as a result, the reaction is generated by cytotoxicmolecules, such as perforin and granzyme, released from the NK cell(Non-patent References 16 and 17). The grade of the ADCC activity isgenerally in order of IgG1>IgG3>>IgG4≧IgG2 (Non-patent References 18 and19).

The CDC activity is a reaction in which an antibody bound to its antigenactivates reaction cascade of a group of serum proteins, called serumcomplement system, and finally lyses the target cell. The CDC activityis high in human IgG1 and IgG3, and the grade is generally in order ofIgG3≧IgG1>>IgG 2≈IgG4. The complement system is classified intorespective components of C1 to C9, and most of them are enzymeprecursors which express enzyme activities by partial degradation. TheCDC activity starts with the binding of C1q as a component of C1 to theFc region of an antibody on the target cell, each of the subsequentcomponents is partially degraded by the former step component to advancecascade of the activation, and finally, C5 to C9 form a pore-formingpolymer called membrane attacking complex on the cell membrane of thetarget cell to cause the cell lysis reaction (Non-patent References 16and 17).

Importance of the above-described effector functions is also recognizedon the mechanism of action of therapeutic antibodies used in theclinical field. The above-described Rituxan™ is a human chimericantibody of IgG1 subclass, and not only it shows ADCC activity and CDCactivity in vitro (Non-patent Reference 21) but it has also beensuggested on its clinical effects that Rituxan™ actually exerts effectorfunctions in the body of patients, because of the facts that itstherapeutic affect is high in the patients showing a genotype of strongADCC activity (Non-patent Reference 22), that the complement componentsare quickly consumed from blood after its administration (Non-patentReference 23), and that expression of CD59 as a factor for suppressingCDC activity increases in cancer cells of relapsed patients after itsadministration (Non-patent Reference 24). Herceptin™ is also a humanizedantibody of IgG1 subclass, and it has been reported that it has ADCCactivity in vitro (Non-patent Reference 25).

Based on the above, human IgG1 antibodies are most suitable astherapeutic antibodies, because they have higher ADCC activity and CDCactivity and also have longer half-life in human blood than othersubclasses.

In order to analyze functions of IgG antibodies, studies have beencarried out for the preparation of antibodies in which the domain unitswere swapped among different IgG subclasses. In the latter half of1980s, Morrison et al. have pointed out that antibody molecules in whichrespective domains (CH1, CH2, CH3, hinge) of the heavy chain constantregion were swapped between IgG1 and IgG4, or between IgG2 and IgG3, canbe expressed as recombinant proteins, and that antibodies in which thehinges of IgG3 and IgG4 were mutually swapped do not show changes in therespective complement fixation capacity and Fc receptor binding abilityof the original antibodies (Patent Reference 1). Thereafter, they haveexamined these domain-swapped antibodies of IgG1 with IgG4 and IgG2 withIgG3 and shown as a result that the C-terminal side of CH2 is importantfor the CDC activity of IgG1, and CH2 for the CDC activity of IgG3(Non-patent Reference 26), and that the CH2 domain and hinge areimportant for the binding of IgG1 and IgG3 to FcγRI which is one of theFc receptors (Non-patent Reference 27).

It is known that C1q binds to the Fc region of antibody molecules.Binding constants (Ka) of C1q for monomers of human IgG1, IgG2, IgG3 andIgG4 are 1.2×10⁴, 0.64×10⁴, 2.9×10⁴ and 0.44×10⁴ M⁻¹, respectively(Non-patent Reference 20). As described in the above, the CH2 domain isparticularly important in the Fc region (Non-patent Reference 26), andmore illustratively, it is known that, according to the definition of EUindex by Kabat et al. (Non-patent Reference 28), Leu 235 (Non-patentReference 29) and Asp 270, Lys 322, Pro 329 and Pro 331 (Non-patentReference 30) in the CH2 are important in the case of human IgG1, andGlu 233, Leu 234, Leu 235 and Gly 236 (Non-patent Reference 31) and Lys322 (Non-patent Reference 32) in the case of human IgG3.

Attempts have been made to further enhance CDC activity by replacing apart of the amino acid sequence of heavy chain constant region of humanIgG3, as a subclass having the highest CDC activity, by an amino acidsequence from other subclass. Regarding hinge lengths of respective IgGsubclasses, IgG1 has 15 amino acids, IgG2 has 12 amino acids, IgG3 has62 amino acids, and IgG4 has 12 amino acids, so that the human IgG3 hasa structural characteristic that its hinge region is longer than otherIgG subclasses (Non-patent Reference 1). Michaelsen et al. have pointedout that CDC activity of an Ig, in which the 62 amino acids of the hingeof wild type human IgG3 polypeptide were shortened to 15 amino acids bydeleting 3 exons of the N-terminal side, exceeds those of IgG3 and IgG1(Non-patent Reference 33). In addition, Norderhaug et al. have pointedout that the CDC activity is further enhanced when the amino acidsequence of the above-described shortened hinge is allowed toapproximate the amino acid sequence of the hinge of IgG4 (Non-patentReference 34). Also, Brekke et al. have pointed out that in an IgG3 inwhich its hinge part is replaced by IgG1, and the hinge part, and anIgG3 in which an N-terminal moiety of CH1 is replaced by IgG1, the CDCactivity is higher than that of IgG3 and becomes equal to or higher thanthat of IgG1 (Non-patent Reference 35).

In addition, attempts have also been made to enhance the CDC activity bypreparing modified forms of IgG by introducing mutation in all sorts ofamino acid sequence in the human IgG heavy chain constant regions, andincreasing the binding activity of these modified forms to C1q. Idusogieet al. have reported that the CDC activity is enhanced approximately by2-fold at the maximum by replacing when Lys at position 326 or Glu atposition 333 indicated by the EU index in the CH2 domain in the heavychain constant region of an anti-CD20 chimeric antibody Rituxan™ havinghuman IgG1 constant region and mouse-derived variable region is replacedby other amino acid (Non-patent Reference 36, Patent Reference 2).Idusogie et al. also have pointed out that the CDC activity of IgG2,which is approximately one-to-several hundreds of the CDC activity ofIgG1, increases to about 1/25 of the CDC activity of IgG1, when Lys atposition 326 or Glu at position 333 indicated by the EU index isreplaced by other amino acid (Patent References 3 to 5).

FcγR-dependent activity such as ADCC activity or phagocytotic activityand CDC activity are both important for the therapeutic effect oftherapeutic antibodies. However, since both of the C1q binding as theearly stage for inducing CDC activity and the binding to FcγR as theearly stage for inducing ADCC activity mediate the antibody Fc, there isa possibility that the ADCC activity is reduced when the CDC activity isenhanced. Idusogie et al. have reported that a point mutation-introducedmutant of Fc amino acids of CDC activity-enhanced IgG shows sharplyreduced ADCC activity (Non-patent Reference 36).

Also, it is known that the ADCC activity of an antibody having a humanIgG constant region changes by the structure of the complex typeN-glycoside-linked sugar chain (its schematic illustration is shown inFIG. 2) to be added to asparagine at position 297 in the CH2 domain(Patent Reference 6). Although there are reports stating that the ADCCactivity of antibodies changes depending on the contents of galactoseand N-acetylglucosamine in the sugar chain to be bound to the antibody(Non-patent References 37 to 40), the substance which mostly influenceson the ADCC activity is a fucose bound to N-acetylglucosamine in thereducing terminal through α1,6 bond. An IgG antibody having a complextype N-glycoside-linked sugar chain in which fucose does not bind toN-acetylglucosamine in the reducing terminal shows remarkably higherADCC activity than that of an IgG antibody having a complex typeN-glycoside-linked sugar chain in which fucose is bound toN-acetylglucosamine in the reducing terminal (Non-patent References 41and 42, Patent Reference 7). Cells in which the α1,6-fucosyltransferasegene was knocked out are known as the cell which produces an antibodycomposition having a complex type N-glycoside-linked sugar chain inwhich fucose is not bound to N-acetylglucosamine in the reducingterminal (Patent References 7 and 8).

Since human IgG3 does not have binding activity to protein A unlikeother subclasses (Non-patent Reference 1), it is difficult to purify itwhen produced as a medicine. It is known that IgG molecules associatewith protein A at the interface of CH2 domain and CH3 domain,illustratively, it has been suggested based on an X-ray crystallographythat a loop moiety containing amino acids of positions 252 to 254 andpositions 308 to 312 indicated by the EU index in the immunoglobulinstructure (immunoglobulin fold) of CH2 and positions 433 to 436 in theimmunoglobulin structure of CH3 is important (Non-patent Reference 43).It was further shown by a nuclear magnetic resonance method (NMR method)that Ile 253, Ser 254, His 310 and Gln 311 in the CH2 and His 433, His435 and His 436 in the CH3 of IgG 1 are important (Non-patent Reference44). In addition, Kim et al. have found that the binding activity toprotein A is decreased when His 435 in the human IgG1 heavy chainconstant region is replaced with Arg derived from IgG3 (Non-patentReference 45).

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SUMMARY OF THE INVENTION

An antibody which does not have antigenicity, has enhanced effectorfunctions such as CDC activity and ADCC activity and has improvedtherapeutic effect is in demand. In addition, an antibody which can beproduced as a medicine is in demand.

The present invention provides a recombinant antibody composition havinghigher complement-dependent cytotoxic activity than a human IgG1antibody and a human IgG3 antibody, wherein a polypeptide comprising aCH2 domain in the Fc region of a human IgG1 antibody is replaced by apolypeptide comprising an amino acid sequence which corresponds to thesame position of a human IgG3 antibody indicated by the EU index as inKabat, et al.; a DNA encoding the antibody molecule or a heavy chainconstant region of the antibody molecule contained in the recombinantantibody composition; a transformant obtainable by introducing therecombinant vector into a host cell; a process for producing therecombinant antibody composition using the transformant; and amedicament comprising the recombinant antibody composition as an activeingredient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows amino acid sequences of heavy chain constant regions ofrespective IgG subclasses.

FIG. 2 is a schematic illustration showing structure of a complex typeN-linked sugar chain bound to asparagine at position 297 in the H chainof IgG antibody.

FIG. 3 shows construction steps of a plasmid pKANTEX2B8γ3.

FIG. 4 is a schematic illustration showing an anti-CD20 domain-swappedantibody.

FIG. 5 shows a plasmid pKTX93/1133.

FIG. 6 shows a plasmid pKTX93/3311.

FIG. 7 shows binding activity of various CD20 domain-swapped antibodies,an anti-CD20 human IgG1 chimeric antibody and an anti-CD20 human IgG3chimeric antibody with an anti-CD20 antibody CD20-IgG1(+F) in acompetitive inhibition assay to Daudi cell. The abscissa shows sampleconcentration, and the ordinate shows binding inhibition ratio at eachsample concentration. Δ and ▾ in the graphs are common to graphs A to Dand show a negative control anti-Her2 antibody Herceptin™ (Δ) and ananti-CCR4 antibody KM3060 (▾). Regarding ∘ and  in the graphs, thecorresponding sample is different in each graph, and graph A showsCD20-IgG1(+F) (∘) and CD20-IgG1(−F) (), graph B shows CD20-IgG3(+F) (∘)and CD20-IgG3(−F) (), graph C shows 1133(+F) (∘) and 1133(−F) (), andgraph D shows 3311(+F) (∘) and 3311(−F) ().

FIG. 8 shows CDC activity of anti-CD20 human IgG1 chimeric antibodies,anti-CD20 human IgG3 chimeric antibodies and anti-CD20 domain-swappedantibodies 1133 and 3311 to Daudi cell. The abscissa shows sample names,and the ordinate shows CDC activity. The graph shows CDC activity ofeach sample at a concentration of 0.3 μg/ml.

FIG. 9 shows CDC activity of anti-CD20 human IgG1 chimeric antibodies,anti-CD20 human IgG3 chimeric antibodies and 1133-type anti-CD20domain-swapped antibodies to ST 486 cell (A) or Raji cell (B). Theabscissa shows sample concentration, and the ordinate shows CDC activityin each sample concentration. In the graph, □ shows CD20-IgG1(+F),▪shows CD20-IgG1(−F), Δ shows CD20-IgG3(+F), ▾ shows CD20-IgG3(−F), ∘shows 1133(+F) and  shows 1133(−F).

FIG. 10 shows ADCC activity of anti-CD20 human IgG1 chimeric antibodies,anti-CD20 human IgG3 chimeric antibodies, 1133-type anti-CD20domain-swapped antibodies and 3311-type anti-CD20 domain-swappedantibodies to Daudi cell. The abscissa shows sample concentration, andthe ordinate shows ADCC activity at each sample concentration. Regarding∘ and  in the graphs, the corresponding sample is different in eachgraph, and graph A shows CD20-IgG1(+F) (∘) and CD20-IgG1(−F) (), graphB shows CD20-IgG3(+F) (∘) and CD20-IgG3(−F) (), graph C shows 1133(+F)(∘) and 1133(−F) (), and graph D shows 3311(+F) (∘) and 3311(−F) ().

FIG. 11 shows binding activity of 1133-type anti-CD20 domain-swappedantibody, anti-CD20 human IgG1 chimeric antibody and anti-CD20 humanIgG3 chimeric antibody to soluble human FcγRIIa (valine type) (A to C)or soluble human FcγRIIa (phenylalanine type) (D to F), in ELISA in theabsence of the antigen CD20. The abscissa shows sample concentration,and the ordinate shows absorbance at each sample concentration. Graphs Aand D show binding activity of CD20-IgG1(−F) () and CD20-IgG1(+F) (∘),graphs B and E show that of CD20-IgG3(−F) () and CD20-IgG3(+F) (∘), andgraphs C and F show that of 1133(−F) () and 1133(+F) (∘).

FIG. 12 is a schematic illustration showing an anti-CD20 domain-swappedantibody.

FIG. 13 shows a plasmid pKANTEX2B8P.

FIG. 14 shows positions of the restriction enzyme recognition sites ApaIand SmaI of a plasmid pKANTEX93/1133.

FIG. 15 shows a plasmid pKANTEX93/1113.

FIG. 16 shows a plasmid pKANTEX93/1131.

FIG. 17 shows SDS-PAGE electrophoresis patterns of purified 1133-typeanti-CD20 domain-swapped antibody, 1113-type anti-CD20 domain-swappedantibody, 1131-type anti-CD20 domain-swapped antibody, anti-CD20 humanIgG1 chimeric antibody CD20-IgG1 and anti-CD20 human IgG3 chimericantibody CD20-IgG3. Staining of protein was carried out with CoomassieBrilliant Blue (CBB). Lane 1 corresponds to CD20-IgG1, lane 2corresponds to CD20-IgG3, lane 3 corresponds to 1133 and lane 4corresponds to 1113.

FIG. 18 shows CDC activity of 1133-type anti-CD20 domain-swappedantibody, 1113-type anti-CD20 domain-swapped antibody, 1131-typeanti-CD20 domain-swapped antibody, human IgG1 anti-CD20 antibodyCD20-IgG1 and human IgG3 anti-CD20 antibody CD20-IgG3 to ST 486 cell (A)or Raji cell (B). The abscissa shows sample concentration, and theordinate shows cytotoxicity ratio at each sample concentration. In thegraph, ▪ shows CD20-IgG1, ▾ shows CD20-IgG3,  shows 1133, × shows 1113and ♦ shows 1131.

FIG. 19 shows ADCC activity of 1133-type anti-CD20 domain-swappedantibody, 1113-type anti-CD20 domain-swapped antibody, 1131-typeanti-CD20 domain-swapped antibody, anti-CD20 human IgG1 chimericantibody CD20-IgG1 and anti-CD20 human IgG3 chimeric antibody CD20-IgG3to Daudi cell. The abscissa shows sample concentration, and the ordinateshows ratio of cytotoxicity at each sample concentration. In the graph,▪ shows CD20-IgG1, ▾ shows CD20-IgG3,  shows 1133, × shows 1113 and ♦shows 1131.

FIG. 20 shows a result of the measurement by ELISA assay, of the bindingactivity of anti-CD20 human IgG1 chimeric antibodies CD20-IgG1(−F) andCD20-IgG1(+F) and 1133-type anti-CD20 domain-swapped antibody 1133(−F)and 1133(+F) to an Fc receptor family FcγRI (A) or FcγRIIa (B). Theabscissa shows sample concentration, and the ordinate shows absorbanceat each sample concentration. Graph A shows binding activities ofCD20-IgG1(−F) (▾), CD20-IgG1(+F) (Δ), 1133(−F) () and 1133(+F) (∘) toFcRI, and group B shows those to FcγRIIa.

FIG. 21 is a schematic illustration showing domain structures ofantibodies 113A, 113B, 113C, 113D, 113E, 113F, 113G and 113H prepared bypartially replacing the CH3 domain of 1133-type anti-CD20 domain-swappedantibody with a human IgG1 sequence. In the drawing, the regionrepresented by □ shows amino acid sequence of IgG1, and the regionrepresented by ▪ shows amino acid sequence of IgG3, and the numeralsshown on the upper side of both terminals of the IgG3 region are EUindexes which correspond to the positions of the IgG3 amino acidresidues positioned on both terminals.

FIG. 22 shows construction steps of expression vector plasmid of variousantibodies in which the CH3 domain of 1133-type anti-CD20 domain-swappedantibody was partially replaced by a human IgG1 sequence.

FIG. 23 shows SDS-PAGE electrophoresis patterns of purified samples inwhich the CH3 domain of 1133-type anti-CD20 domain-swapped antibody waspartially replaced by a human IgG1 sequence. Staining of protein wascarried out with Coomassie Brilliant Blue (CBB). Starting from the leftside, the lanes corresponds to molecular weight markers, CD20-IgG1(−F),1133(−F), 113A(−F), 113B(−F), 113C(−F), 113D(−F), 113E(−F), 113F(−F),113G(−F), and 113H(−F).

FIG. 24 shows CDC activity of various antibodies in which the CH3 domainof 1133-type anti-CD20 domain-swapped antibody was partially replaced bya human IgG1 sequence, 1133-type anti-CD20 domain-swapped antibody and1131-type anti-CD20 domain-swapped antibody to CD20-positive cells. Theabscissa shows sample concentration, and the ordinate shows CDC activityat each sample concentration. In the drawings,  (thick line) shows1133(−F), ∘ (thick line) shows 1131(−F),  (thin line) shows 113A(−F), ∘(thin line) shows 113B(−F), ▾ shows 113C(−F), Δ shows 113D(−F), ♦ shows113E(−F), ⋄ shows 113F(−F), ▪ shows 113G(−F) and □ shows 113H(−F).

FIG. 25 shows a result of the measurement, by ELISA system, of thebinding activity of various antibodies in which the CH3 domain of1133-type anti-CD20 domain-swapped antibody was partially replaced by ahuman IgG1 sequence, an anti-CD20 human IgG1 chimeric antibodyCD20-IgG1, an anti-CD20 human IgG3 chimeric antibody CD20-IgG3 and1133-type, 1131-type and 1113-type anti-CD20 domain-swapped antibodiesto protein A. The abscissa shows sample concentration, and the ordinateshows absorbance at each sample concentration. FIG. 25A shows bindingactivity of CD20-IgG1(−F) (), CD20-IgG3(−F) (∘), 1133(−F) (▪), 1131(−F)(□) and 1113(−F) (Δ) to protein A. FIG. 25B shows binding activity ofCD20-IgG1(−F) (), 1133(−F) (▪), 113A(−F) (∘), 113B(−F) (□), 113C(−F)(+), 113D(−F) (*), 113E(−F) (⋄), 113F(−F) (♦), 113G(−F) (▾) and 113H(−F)(Δ) to protein A.

FIG. 26 shows CDC activity of an anti-CD20 human IgG1 chimeric antibodyCD20-IgG1 and 1133-type, 1131-type and 113F-type anti-CD20domain-swapped antibodies to a CD20-positive CLL cell line MEC-1 (A),MEC-2 (B) or EHEB (C). The abscissa shows sample concentration, and theordinate shows CDC activity at each sample concentration. In thedrawing, ∘ shows CD20-IgG1,  shows 1133, Δ shows 1131 and ▾ shows 113F,respectively.

FIG. 27 shows construction steps of an expression vector plasmid of1133-type anti-Campath domain-swapped antibody.

FIG. 28 shows construction steps of an expression vector plasmid ofanti-Campath human IgG1 antibody.

FIG. 29 shows construction steps of an expression vector plasmid of1131-type anti-Campath domain-swapped antibody.

DETAILED DESCRIPTION OF THE INVENTION

Specifically, the present invention relates to the following (1) to(25):

-   (1) A recombinant antibody composition having higher    complement-dependent cytotoxic activity than a human IgG1 antibody    and a human IgG3 antibody, wherein a polypeptide comprising a CH2    domain in the Fc region of a human IgG1 antibody is replaced by a    polypeptide comprising an amino acid sequence which corresponds to    the same position of a human IgG3 antibody indicated by the EU index    as in Kabat, et al. (hereinafter referred to as EU index).-   (2) The recombinant antibody composition according to (1), further    having binding activity to protein A, which is substantially equal    to that of a human IgG1 antibody.-   (3) The recombinant antibody composition according to (1), wherein    the polypeptide comprising a CH2 domain in the Fc region of a human    IgG1 antibody to be replaced is a polypeptide selected from the    following 1 to 10:-   1. a polypeptide comprising the amino acid sequence at positions 231    to 340 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    80);-   2. a polypeptide comprising the amino acid sequence at positions 231    to 356 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    81);-   3. a polypeptide comprising the amino acid sequence at positions 231    to 358 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    82);-   4. a polypeptide comprising the amino acid sequence at positions 231    to 384 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    83);-   5. a polypeptide comprising the amino acid sequence at positions 231    to 392 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    84);-   6. a polypeptide comprising the amino acid sequence at positions 231    to 397 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    85);-   7. a polypeptide comprising the amino acid sequence at positions 231    to 422 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    86);-   8. a polypeptide comprising the amino acid sequences at positions    231 to 434 (SEQ ID NO: 87)) and at positions 436 to 447 (SEQ ID    NO: 88) of an IgG1 antibody indicated by the EU index;-   9. a polypeptide comprising the amino acid sequence at positions 231    to 435 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    89); and-   10. a polypeptide comprising the amino acid sequence at positions    231 to 447 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    90).-   (4) The recombinant antibody composition according to (2), wherein    the polypeptide comprising a CH2 domain in the Fc region of a human    IgG1 antibody to be replaced is a polypeptide selected from the    following 1 to 8:-   1. a polypeptide comprising the amino acid sequence at positions 231    to 340 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    80);-   2. a polypeptide comprising the amino acid sequence at positions 231    to 356 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    81);-   3. a polypeptide comprising the amino acid sequence at positions 231    to 358 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    82);-   4. a polypeptide comprising the amino acid sequence at positions 231    to 384 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    83);-   5. a polypeptide comprising the amino acid sequence at positions 231    to 392 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    84);-   6. a polypeptide comprising the amino acid sequence at positions 231    to 397 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    85);-   7. a polypeptide comprising the amino acid sequence at positions 231    to 422 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    86); and-   8. a polypeptide comprising the amino acid sequences at positions    231 to 434 (SEQ ID NO: 87) and at positions 436 to 447 (SEQ ID    NO: 88) of an IgG1 antibody indicated by the EU index.-   (5) The recombinant antibody composition according to any one of (1)    to (4), comprising an antibody molecule having complex type    N-glycoside-linked sugar chains in the Fc region, wherein the ratio    of sugar chains in which fucose is not bound to N-acetylglucosamine    in the reducing terminal of the sugar chains among the total complex    type N-glycoside-linked sugar chains which bind to the Fc region    contained in the composition is 20% or more.-   (6) The recombinant antibody composition according to any one of (1)    to (4), comprising an antibody molecule having complex type    N-glycoside-linked sugar chains in the Fc region, wherein the    complex type N-glycoside-linked sugar chains bound to the Fc region    of the antibody are sugar chains in which fucose is not bound to    N-acetylglucosamine in the reducing terminal in the sugar chains.-   (7) A DNA encoding the antibody molecule contained in the    recombinant antibody composition described in any one of (1) to (4).-   (8) A DNA encoding a heavy chain constant region of the antibody    molecule contained in the recombinant antibody composition described    in any one of (1) to (4).-   (9) A transformant obtainable by introducing the DNA described    in (8) into a host cell.-   (10) The transformant according to (9), wherein the host cell is a    cell resistant to a lectin which recognizes a sugar chain structure    in which 1-position of fucose is bound to 6-position of    N-acetylglucosamine in the reducing terminal through α-bond in the    N-glycoside-linked sugar chain.-   (11) The transformant according to (9), wherein when a gene encoding    an antibody molecule is introduced into the host cell, the host cell    is capable of producing an antibody composition comprising an    antibody molecule having complex type N-glycoside-linked sugar    chains in the Fc region, wherein the ratio of sugar chains in which    fucose is not bound to N-acetylglucosamine in the reducing terminal    of the sugar chains among the total complex type N-glycoside-linked    sugar chains which bind to the Fc region contained in the    composition is 20% or more.-   (12) The transformant according to (11), wherein the sugar chains in    which fucose is not bound are sugar chains in which 1-position of    fucose is bound to 6-position of N-acetylglucosamine in the reducing    terminal through α-bond in the complex type N-glycoside-linked sugar    chain.-   (13) The transformant according to (9), wherein the host cell is a    cell in which a genome is modified so as to have decreased or    deleted activity of an enzyme relating to the synthesis of an    intracellular sugar nucleotide, GDP-fucose and/or an enzyme relating    to the modification of a sugar chain in which 1-position of fucose    is bound to 6-position of N-acetylglucosamine in the reducing    terminal through α-bond in the complex type N-glycoside-linked sugar    chain.-   (14) The transformant according to (9), wherein the host cell is a    cell in which all of alleles on a genome encoding an enzyme relating    to the synthesis of an intracellular sugar nucleotide, GDP-fucose    and/or an enzyme relating to the modification of a sugar chain in    which 1-position of fucose is bound to 6-position of    N-acetylglucosamine in the reducing terminal through α-bond in the    complex type N-glycoside-linked sugar chain are knocked out.-   (15) The transformant according to (13) or (14), wherein the enzyme    relating to the synthesis of an intracellular sugar nucleotide,    GDP-fucose is an enzyme selected from GDP-mannose 4,6-dehydratase    (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase (Fx).-   (16) The transformant according to (15), wherein the GDP-mannose    4,6-dehydratase is a protein encoded by a DNA selected from the    group consisting of the following (a) and (b):-   (a) a DNA comprising the nucleotide sequence represented by SEQ ID    NO:18;-   (b) a DNA which hybridizes with the DNA consisting of the nucleotide    sequence represented by SEQ ID NO:18 under stringent conditions and    encodes a protein having GDP-mannose 4,6-dehydratase activity.-   (17) The transformant according to (15), wherein the GDP-mannose    4,6-dehydratase is a protein selected from the group consisting of    the following (a) to (c):-   (a) a protein comprising the amino acid sequence represented by SEQ    ID NO:19;-   (b) a protein consisting of an amino acid sequence in which one or    more amino acid(s) is/are deleted, substituted, inserted and/or    added in the amino acid sequence represented by SEQ ID NO:19 and    having GDP-mannose 4,6-dehydratase activity;-   (c) a protein consisting of an amino acid sequence which has 80% or    more homology with the amino acid sequence represented by SEQ ID    NO:19 and having GDP-mannose 4,6-dehydratase activity.-   (18) The transformant according to (15), wherein the    GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase is a protein encoded by a    DNA selected from the group consisting of the following (a) and (b):-   (a) a DNA comprising the nucleotide sequence represented by SEQ ID    NO:20;-   (b) a DNA which hybridizes with the DNA consisting of the nucleotide    sequence represented by SEQ ID NO:20 under stringent conditions and    encodes a protein having GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase    activity.-   (19) The transformant according to (16), wherein the    GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase is a protein selected    from the group consisting of the following (a) to (c):-   (a) a protein comprising the amino acid sequence represented by SEQ    ID NO:21;-   (b) a protein consisting of an amino acid sequence in which one or    more amino acid(s) is/are deleted, substituted, inserted and/or    added in the amino acid sequence represented by SEQ ID NO:21 and    having GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity;-   (c) a protein consisting of an amino acid sequence which has 80% or    more homology with the amino acid sequence represented by SEQ ID    NO:21 and has GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity.-   (20) The transformant according to (13) or (14), wherein the enzyme    relating to the modification of a sugar chain in which 1-position of    fucose is bound to 6-position of N-acetylglucosamine in the reducing    terminal through α-bond in the complex type N-glycoside-linked sugar    chain is α1,6-fucosyltransferase.-   (21) The transformant according to (20), wherein the    α1,6-fucosyltransferase is a protein encoded by a DNA selected from    the group consisting of the following (a) to (d):-   (a) a DNA comprising the nucleotide sequence represented by SEQ ID    NO:22;-   (b) a DNA comprising the nucleotide sequence represented by SEQ ID    NO:23;-   (c) a DNA which hybridizes with the DNA consisting of the nucleotide    sequence represented by SEQ ID NO:22 under stringent conditions and    encodes a protein having α1,6-fucosyltransferase activity;-   (d) a DNA which hybridizes with the DNA consisting of the nucleotide    sequence represented by SEQ ID NO:23 under stringent conditions and    encodes a protein having α-1,6-fucosyltransferase activity.-   (22) The transformant according to (20), wherein the    α1,6-fucosyltransferase is a protein selected from the group    consisting of the following (a) to (f):-   (a) a protein comprising the amino acid sequence represented by SEQ    ID NO:24;-   (b) a protein comprising the amino acid sequence represented by SEQ    ID NO:25;-   (c) a protein consisting of an amino acid sequence in which one or    more amino acid(s) is/are deleted, substituted, inserted and/or    added in the amino acid sequence represented by SEQ ID NO:24 and    having α1,6-fucosyltransferase activity;-   (d) a protein consisting of an amino acid sequence in which one or    more amino acid(s) is/are deleted, substituted, inserted and/or    added in the amino acid sequence represented by SEQ ID NO:25 and    having α1,6-fucosyltransferase activity;-   (e) a protein consisting of an amino acid sequence which has 80% or    more homology with the amino acid sequence represented by SEQ ID    NO:24 and having α1,6-fucosyltransferase activity;-   (f) a protein consisting of an amino acid sequence which has 80% or    more homology with the amino acid sequence represented by SEQ ID    NO:25 and having α1,6-fucosyltransferase activity.-   (23) The transformant according to any one of (9) to (22), wherein    the host cell is a cell selected from the group consisting of the    following (a) to (i):-   (a) a CHO cell derived from a Chinese hamster ovary tissue;-   (b) a rat myeloma cell line, YB2/3HL.P2.G11.16Ag.20 cell;-   (c) a mouse myeloma cell line, NS0 cell;-   (d) a mouse myeloma cell line, 5P2/0-Ag14 cell;-   (e) a BHK cell derived from a syrian hamster kidney tissue;-   (f) an antibody-producing hybridoma cell;-   (g) a human leukemia cell line, Namalwa cell;-   (h) an embryonic stem cell;-   (i) a fertilized egg cell.-   (24) A process for producing a recombinant antibody composition,    which comprises culturing the transformant described in any one    of (9) to (23) in a medium to form and accumulate the antibody    composition in the culture; and recovering and purifying the    antibody composition from the culture.-   (25) A medicament comprising the recombinant antibody composition    described in any one of (1) to (6) as an active ingredient.

An antibody molecule is constituted by polypeptides called H chain and Lchain. Also, the H chain is constituted by regions of a variable region(VH) and CH from its N-terminal, and the L chain is constituted byregions of a variable region (VL) and CL from its N-terminal. CH isfurther constituted by domains of a CH1 domain, a hinge domain, a CH2domain and a CH3 domain. The domain means a functional constitution unitconstituting each polypeptide in the antibody molecule. Also, the CH2domain and the CH3 domain in combination are called Fc region.

The CH1 domain, the hinge domain, the CH2 domain, the CH3 domain and theFc region are defined by positions of amino acid residues from theN-terminal indicated by the EU index as in Kabat, et al. [Sequence ofProteins of Immunological Interest, 5th Edition (1991)]. Specifically,CH1 is defined as the amino acid sequence of positions 118 to 215indicated by the EU index, the hinge is defined as the amino acidsequence of positions 216 to 230 indicated by the EU index, CH2 isdefined as the amino acid sequence of positions 231 to 340 indicated bythe EU index, and CH3 is defined as the amino acid sequence of positions341 to 447 indicated by the EU index.

The recombinant antibody composition of the present invention may be anyantibody composition, so long as it is a recombinant antibodycomposition having higher complement-dependent cytotoxic activity than ahuman IgG1 antibody and a human IgG3 antibody, wherein a polypeptidecomprising a CH2 domain in the Fg region of a human IgG1 antibody isreplaced by a polypeptide comprising an amino acid sequence whichcorresponds to the same position of a human IgG3 antibody indicated bythe EU index as in Kabat et al., among the recombinant antibodycompositions in which domains of CH1, the hinge, CH2 and CH3 in theheavy chain constant region of a human IgG1 are swapped into domainscorresponding to IgG3 (hereinafter referred to as domain-swappedantibody).

The recombinant antibody composition of the present invention may be anyantibody composition, so long as it is a fusion protein having a heavychain constant region, having an antibody or heavy chain constant regionwhich has binding activity to a target molecule, and having bindingactivity to a target molecule.

The antibody having binding activity to a target molecule includes ahuman chimeric antibody, a humanized antibody and a human antibody.

The fusion protein having a heavy chain constant region and bindingactivity to a target molecule includes, in the case where the targetmolecule is a ligand, a fusion protein of a receptor for the ligand anda heavy chain constant region; in the case where the target molecule isa receptor, a fusion protein of a ligand for the receptor and a heavychain constant region; a fusion protein of an antibody or antibodyfragment having binding activity to a target molecule and a heavy chainconstant region; and the like.

A human chimeric antibody is an antibody which comprises VH and VL of anon-human animal antibody, and CH and CL of human antibody. Thenon-human animal may be any animal such as a mouse, a rat, a hamster ora rabbit, so long as a hybridoma can be prepared therefrom.

The human chimeric antibody of the present invention can be produced byobtaining cDNAs encoding VH and VL from a monoclonal antibody-producinghybridoma, inserting them into an expression vector for animal cellcomprising DNAs encoding CH and CL of human antibody to therebyconstruct a human chimeric antibody expression vector, and thenintroducing the vector into an animal cell to express the antibody.

As the CH of human chimeric antibody, any CH can be used, so long as itbelongs to human immunoglobulin (hIg), and those belonging to the hIgGclass are preferred, and any one of the subclasses belonging to the hIgGclass, such as γ1, γ2, γ3 and γ4, can be used. As the CL of humanchimeric antibody, any CL can be used, so long as it belongs to the hIgclass, and those belonging to the K class or X class can be used.

A humanized antibody is an antibody in which amino acid sequences ofCDRs of VH and VL of a non-human animal antibody are grafted intoappropriate positions of VH and VL of a human antibody.

The humanized antibody of the present invention can be produced byconstructing cDNAs encoding V regions in which the amino acid sequencesof CDRs of VH and VL of a non-human animal antibody are grafted into theFRs of VH and VL of any human antibody, inserting them into anexpression vector for animal cell comprising DNAs encoding CH and CL ofa human antibody to thereby construct a humanized antibody expressionvector, and then introducing the expression vector into an animal cellto express the humanized antibody.

As the CH of the humanized antibody, any CH can be used, so long as itbelongs to the hIg, and those of the hIgG class are preferred and anyone of the subclasses belonging to the hIgG class, such as γ1, γ2, γ3and γ4, can be used. As the CL of the human CDR-grafted antibody, any CLcan be used, so long as it belongs to the hIg class, and those belongingto the κ class or λ class can be used.

A human antibody is originally an antibody naturally existing in thehuman body, but it also includes antibodies obtained from a humanantibody phage library or a human antibody-producing transgenic animal,which is prepared based on the recent advance in genetic engineering,cell engineering and developmental engineering techniques.

The antibody existing in the human body can be prepared, for example byisolating a human peripheral blood lymphocyte, immortalizing it byinfecting with EB virus or the like and then cloning it to therebyobtain lymphocytes capable of producing the antibody, culturing thelymphocytes thus obtained, and purifying the antibody from the culture.

The human antibody phage library is a library in which antibodyfragments such as Fab and scFv are expressed on the phage surface byinserting a gene encoding an antibody prepared from a human B cell intoa phage gene. A phage expressing an antibody fragment having the desiredantigen binding activity can be recovered from the library, using itsactivity to bind to an antigen-immobilized substrate as the index. Theantibody fragment can be converted further into a human antibodymolecule comprising two full H chains and two full L chains by geneticengineering techniques.

A human antibody-producing transgenic animal is an animal in which ahuman antibody gene is integrated into cells. Specifically, a humanantibody-producing transgenic animal can be prepared by introducing agene encoding a human antibody into a mouse ES cell, grafting the EScell into an early stage embryo of other mouse and then developing it. Ahuman antibody is prepared from the human antibody-producing transgenicnon-human animal by obtaining a human antibody-producing hybridoma by ahybridoma preparation method usually carried out in non-human mammals,culturing the obtained hybridoma and forming and accumulating the humanantibody in the culture.

The antibody fragment having binding activity to a target moleculeincludes Fab, Fab′, F(ab′)₂, scFv, diabody, dsFv, a peptide comprisingCDR, and the like.

An Fab is an antibody fragment having a molecular weight of about 50,000and having antigen binding activity, in which about a half of theN-terminal side of H chain and the entire L chain, among fragmentsobtained by treating IgG with a protease, papain (cleaving an amino acidresidue at the 224th position of the H chain), are bound togetherthrough a disulfide bond (S—S bond).

An F(ab′)₂ is an antibody fragment having antigen binding activity andhaving a molecular weight of about 100,000 which is somewhat larger thanone in which Fab are bound via an S—S bond in the hinge region, amongfragments obtained by treating IgG with a protease, pepsin (by cleavingthe H chain at the 234th amino acid residue).

An Fab′ is an antibody fragment having a molecular weight of about50,000 and having antigen binding activity, which is obtained bycleaving an S—S bond in the hinge region of the F(ab′)₂. An scFv is aVH-P-VL or VL-P-VH polypeptide in which one chain VH and one chain VLare linked using an appropriate peptide linker (P) having 12 or moreresidues and is an antibody fragment having antigen binding activity.

A diabody is an antibody fragment in which scFv's having the same ordifferent antigen binding specificity forms a dimer, and has divalentantigen binding activity to the same antigen or two specific antigenbinding activities to different antigens.

A dsFv is obtained by binding polypeptides in which one amino acidresidue of each of VH and VL is substituted with a cysteine residue viaan S—S bond between the cysteine residues.

A peptide comprising CDR is constituted by including at least one regionor more of CDRs of VH or VL. Plural peptide comprising CDRs can beproduced by binding directly or via an appropriate peptide linker.

Specifically, the recombinant antibody composition of the presentinvention include a recombinant composition in which the polypeptidecomprising a CH2 domain in the Fc region of a human IgG1 antibody to bereplaced is a polypeptide selected from the following 1 to 10:

-   1. a polypeptide comprising the amino acid sequence at positions 231    to 340 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    80);-   2. a polypeptide comprising the amino acid sequence at positions 231    to 356 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    81);-   3. a polypeptide comprising the amino acid sequence at positions 231    to 358 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    82);-   4. a polypeptide comprising the amino acid sequence at positions 231    to 384 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    83);-   5. a polypeptide comprising the amino acid sequence at positions 231    to 392 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    84);-   6. a polypeptide comprising the amino acid sequence at positions 231    to 397 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    85);-   7. a polypeptide comprising the amino acid sequence at positions 231    to 422 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    86);-   8. a polypeptide comprising the amino acid sequences at positions    231 to 434 (SEQ ID NO: 87) and at positions 436 to 447 (SEQ ID    NO: 88) of an IgG1 antibody indicated by the EU index;-   9. a polypeptide comprising the amino acid sequence at positions 231    to 435 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    89); and-   10. a polypeptide comprising the amino acid sequence at positions    231 to 447 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    90).

The amino acid sequence of the CL region in the recombinant antibodycomposition of the present invention may be either an amino acidsequence of a human antibody or an amino acid sequence from a non-humananimal, but it is preferably Cκ or Cλ of an amino acid sequence of ahuman antibody.

In the variable region of the recombinant antibody composition of thepresent invention, VH and VL may be any of an amino acid sequence of ahuman antibody, an amino acid sequence of a non-human animal antibody ora mixed amino acid sequence of these amino acid sequences. Specifically,they include a variable region constituting an antibody produced by ahybridoma, a variable region constituting a humanized antibody, avariable region constituting a human antibody, and the like.

A hybridoma is a cell which is obtained by cell fusion between a B cellobtained by immunizing a non-human mammal with an antigen and a myelomacell derived from mouse or the like and can produce a monoclonalantibody having the desired antigen specificity. Accordingly, thevariable region constituting the antibody produced by the hybridomaconsists of amino acid sequences of non-human animal antibody.

The recombinant antibody composition of the present invention includesantibodies having any specificity, and is preferably an antibody whichrecognizes a tumor-related antigen, an antibody which recognizes anallergy- or inflammation-related antigen, an antibody which recognizescardiovascular disease-related antigen, an antibody which recognizes anautoimmune disease-related antigen or an antibody which recognizes aviral or bacterial infection-related antigen.

The antibody which recognizes a tumor-related antigen includes anti-GD2antibody [Anticancer Res., 13, 331 (1993)], anti-GD3 antibody [CancerImmunol. Immunother., 36, 260 (1993)], anti-GM2 antibody [Cancer Res.,54, 1511 (1994)], anti-HER2 antibody [Proc. Natl. Acad. Sci. USA, 89,4285 (1992)], anti-CD52 antibody [Proc. Natl. Acad. Sci. USA, 89, 4285(1992)], anti-MAGE antibody [British J. Cancer, 83, 493 (2000)],anti-HM1.24 antibody [Molecular Immunol., 36, 387 (1999)],anti-parathyroid hormone-related protein (PTHrP) antibody [Cancer, 88,2909 (2000)], anti-basic fibroblast growth factor antibody,anti-fibroblast growth factor 8 antibody [Proc. Natl. Acad. Sci. USA,86, 9911 (1989)], anti-basic fibroblast growth factor receptor antibody,anti-fibroblast growth factor 8 receptor antibody [J. Biol. Chem., 265,16455 (1990)], anti-insulin-like growth factor antibody [J. Neurosci.Res., 40, 647 (1995)], anti-insulin-like growth factor receptor antibody[J. Neurosci. Res., 40, 647 (1995)], anti-PSMA antibody [J. Urology,160, 2396 (1998)], anti-vascular endothelial cell growth factor antibody[Cancer Res., 57, 4593 (1997)], anti-vascular endothelial cell growthfactor receptor antibody [Oncogene, 19, 2138 (2000)], anti-CD20 antibody[Curr. Opin. Oncol., 10, 548 (1998)], anti-Her2 antibody, anti-CD10antibody, and the like.

The antibody which recognizes an allergy- or inflammation-relatedantigen includes anti-interleukin 6 antibody [Immunol. Rev., 127, 5(1992)], anti-interleukin 6 receptor antibody [Molecular Immunol., 31,371 (1994)], anti-interleukin 5 antibody [Immunol. Rev., 127, 5 (1992)],anti-interleukin 5 receptor antibody, anti-interleukin 4 antibody[Cytokine, 3, 562 (1991)], anti-interleukin 4 receptor antibody [J.Immunol. Meth., 217, 41 (1998)], anti-tumor necrosis factor antibody[Hybridoma, 13, 183 (1994)], anti-tumor necrosis factor receptorantibody [Molecular Pharmacol., 58, 237 (2000)], anti-CCR4 antibody[Nature, 400, 776 (1999)], anti-chemokine antibody [Peri et al., J.Immuno. Meth., 174, 249-257 (1994)], anti-chemokine receptor antibody[J. Exp. Med., 186, 1373 (1997)] or the like. The antibody whichrecognizes a cardiovascular disease-related antigen includesanti-GpIIb/IIIa antibody [J. Immunol., 152, 2968 (1994)],anti-platelet-derived growth factor antibody [Science, 253, 1129(1991)], anti-platelet-derived growth factor receptor antibody [J. Biol.Chem., 272, 17400 (1997)], anti-blood coagulation factor antibody[Circulation, 101, 1158 (2000)] and the like.

The antibody which recognizes a viral or bacterial infection-relatedantigen includes anti-gp120 antibody [Structure, 8, 385 (2000)],anti-CD4 antibody [J. Rheumatology, 25, 2065 (1998)], anti-CCR5 antibodyand anti-Vero toxin antibody [J. Clin. Microbiol., 37, 396 (1999)] andthe like.

The recombinant antibody composition of the present invention has higherCDC activity than a human IgG1 antibody and a human IgG3 antibody byreplacing polypeptide comprising a CH2 domain in the Fc region of ahuman IgG1 antibody with a polypeptide comprising an amino acid sequencewhich corresponds to the same position of a human IgG3 antibodyindicated by the EU index.

Furthermore, the recombinant antibody composition of the presentinvention includes recombinant antibody composition having highercomplement-dependent cytotoxic activity than a human IgG1 antibody and ahuman IgG3 antibody and having binding activity to protein A, which issubstantially equal to that of a human IgG1 antibody, wherein apolypeptide comprising a CH2 domain in the Fc region of a human IgG1antibody is replaced by a polypeptide comprising an amino acid sequencewhich corresponds to the same position of a human IgG3 antibodyindicated by the EU index as in Kabat, et al.

Specifically, examples include the recombinant antibody compositionwherein the polypeptide comprising a CH2 domain in the Fc region of ahuman IgG1 antibody to be replaced is a polypeptide selected from thefollowing 1 to 8:

-   1. a polypeptide comprising the amino acid sequence at positions 231    to 340 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    80);-   2. a polypeptide comprising the amino acid sequence at positions 231    to 356 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    81);-   3. a polypeptide comprising the amino acid sequence at positions 231    to 358 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    82);-   4. a polypeptide comprising the amino acid sequence at positions 231    to 384 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    83);-   5. a polypeptide comprising the amino acid sequence at positions 231    to 392 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    84);-   6. a polypeptide comprising the amino acid sequence at positions 231    to 397 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    85);-   7. a polypeptide comprising the amino acid sequence at positions 231    to 422 of an IgG1 antibody indicated by the EU index (SEQ ID NO:    86);-   8. a polypeptide comprising the amino acid sequences at positions    231 to 434 (SEQ ID NO: 87) and at positions 436 to 447 (SEQ ID    NO: 88) of an IgG1 antibody indicated by the EU index.

The binding activity to protein A can be measured by ELISA, surfaceplasmon resonance or the like. Specifically, the antibody composition isallowed to react with protein A solid-phased on a plate and then isfurther allowed to react with an antibody which recognizes the variouslylabeled antibodies, and the binding activity can be measured bydetermining the antibody composition bound to protein A.

Also, the antibody composition is allowed to react with protein A boundto a carrier such as sepharose at high pH conditions such as a pH ofabout 5 to 8, followed by washing, and then the binding activity can bemeasured by determining the antibody composition eluted at low pHconditions such as a pH of about 2 to 5.

The Fc region in the antibody molecule comprises regions to whichN-glycoside-linked sugar chains are bound. Accordingly, two sugar chainsare bound per one antibody molecule.

The N-glycoside-linked sugar chain include a complex type sugar chain inwhich the non-reducing terminal side of the core structure comprises oneor plurality of parallel side chains of galactose-N-acetylglucosamine(hereinafter referred to as “Gal-GlcNAc”) and the non-reducing terminalside of Gal-GlcNAc further comprises a structure of sialic acid,bisecting N-acetylglucosamine or the like.

In the present invention, the complex type N-glycoside-linked sugarchain is represented by the following formula:

Among the recombinant antibody compositions of the present invention,the recombinant antibody composition comprising an antibody molecule inthe Fc region of the N-glycoside-linked sugar chain may comprise anantibody molecule having the same sugar chain structure or an antibodymolecule having different sugar chain structures, so long as it has theabove sugar chain structure. That is, the recombinant antibodycomposition of the present invention means a composition comprising arecombinant antibody molecule having the same or different sugar chainstructure(s).

Furthermore, among the recombinant antibodies of the present invention,the antibody composition comprising an antibody molecule having complextype N-glycoside-linked sugar chains in the Fc region, wherein the ratioof sugar chains in which fucose is not bound to N-acetylglucosamine inthe reducing terminal of the sugar chains among the total complex typeN-glycoside-linked sugar chains which bind to the Fc region contained inthe composition is 20% or more, has high ADCC activity in addition toCDC activity.

In the present invention, the sugar chain in which fucose is not boundmay have any sugar chain structure in the non-reducing terminal, so longas fucose is not bound to N-acetylglucosamine in the reducing terminalin the above formula.

In the present invention, the case where fucose is not bound toN-acetylglucosamine in the reducing terminal in the sugar chain meansthat fucose is not substantially bound. An antibody composition in whichfucose is not substantially bound specifically refers to an antibodycomposition in which fucose is not substantially detected, i.e., thecontent of fucose is below the detection limit, when subjected to thesugar chain analysis described in the following item 4. A recombinantantibody composition in which fucose is not bound to N-acetylglucosaminein the reducing terminals of all sugar chains has highest ADCC activity.

The ratio of sugar chains in which fucose is not bound toN-acetylglucosamine in the reducing terminal in the sugar chainscontained in the composition which comprises an antibody molecule havingcomplex type N-glycoside-linked sugar chains in the Fc region can bedetermined by releasing the sugar chains from the antibody moleculeusing a known method such as hydrazinolysis or enzyme digestion[Biochemical Experimentation Methods 23—Method for Studying GlycoproteinSugar Chain (Japan Scientific Societies Press), edited by ReikoTakahashi (1989)], carrying out fluorescence labeling or radioisotopelabeling of the released sugar chains and then separating the labeledsugar chains by chromatography. Also, the released sugar chains can alsobe determined by analyzing it with the HPAED-PAD method [J. Liq.Chromatogr., 6, 1577 (1983)].

The transformant producing the recombinant antibody composition of thepresent invention can be obtained by introducing, into an animal cell,an animal cell expression vector into which DNAs encoding a variableregion and a constant region of an antibody molecule are inserted.

The animal cell expression vector is constructed below.

Each of the above DNAs encoding CH and CL is introduced into anexpression vector for animal cell to produce an expression vector foranimal cell.

The expression vector for animal cell includes pAGE107 (JapanesePublished Unexamined Patent Application No. 22979/91; Miyaji H. et al.,Cytotechnology, 3, 133-140 (1990)), pAGE103 (Mizukami T. and Itoh S., J.Biochem., 101, 1307-1310 (1987)), pHSG274 (Brady G. et al., Gene, 27,223-232 (1984)), pKCR (O'Hare K. et al., Proc. Natl. Acad. Sci. USA.,78, 1527-1531 (1981)), pSG1βd2-4 (Miyaji H. et al., Cytotechnology, 4,173-180 (1990)) and the like. The promoter and enhancer used for theexpression vector for animal cell include SV40 early promoter andenhancer (Mizukami T. and Itoh S., J. Biochem., 101, 1307-1310 (1987)),LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana Y. etal., Biochem. Biophys. Res. Commun., 149, 960-968 (1987)),immunoglobulin H chain promoter (Mason J. O. et al., Cell, 41, 479-487(1985)) and ehnancer (Gillies S. D. et al., Cell, 33, 717-728 (1983))and the like.

The vector for expression of recombinant antibody composition may beeither of a type in which genes encoding the H chain and L chain existon separate vectors or of a type in which both genes exist on the samevector (tandem type). In respect of easiness of construction of arecombinant antibody composition expression vector, easiness ofintroduction into animal cells, and balance between the expressionamounts of the H and L chains of an antibody in animal cells, a tandemtype of the vector for expression of recombinant antibody composition ismore preferred (Shitara K. et al., J. Immunol. Methods, 167, 271-278(1994)). The tandem type vector for expression of recombinant antibodycomposition includes pKANTEX93 (WO97/10354), pEE18 (Bentley K. J. etal., Hybridoma, 17, 559-567 (1998)) and the like.

cDNAs encoding VH and VL of antibodies for various antigens are clonedinto the upstream of DNAs encoding CH and CL of the constructed vectorfor expression of recombinant antibody composition to thereby constructa recombinant antibody composition expression vector.

A method for introducing the expression vector into a host cell includeselectroporation (Japanese Published Unexamined Patent Application No.257891-90; Miyaji H. et al., Cytotechnology, 3, 133-140 (1990)) and thelike.

The host cell producing the recombinant antibody composition of thepresent invention may be any host cell which is generally used inproduction of a recombinant protein, such as an animal cell, a plantcell or a microorganism.

The host cell producing the recombinant antibody composition of thepresent invention includes a CHO cell derived from a Chinese hamsterovary tissue, a rat myeloma cell line YB2/3HL.P2.G11.16Ag.20 cell, amouse myeloma cell line NS0 cell, a mouse myeloma SP2/0-Ag14 cell, a BHKcell derived from a syrian hamster kidney tissue, a human leukemia cellline Namalwa cell, a hybridoma cell produced by using a myeloma cell andany B cell, a hybridoma cell produced by a B cell obtained by immunizingwith an antigen a transgenic non-human animal produced by using anembryonic stem cell or a fertilized egg cell and any myeloma cell; ahybridoma cell produced by the above myeloma cell and a B cell obtainedby immunizing a transgenic non-human animal produced by using anembryonic stem cell or a fertilized egg cell; and the like, with anantigen.

The host cell capable of expressing a recombinant antibody compositionhaving high ADCC activity as well as CDC activity includes a host cellresistant to a lectin which recognizes a sugar chain structure in which1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing terminal through α-bond in the complex typeN-glycoside-linked sugar chain, such as a host cell capable of producingan antibody composition comprising an antibody molecule having complextype N-glycoside-linked sugar chains in the Fc region, wherein the ratioof sugar chains in which fucose is not bound to N-acetylglucosamine inthe reducing terminal of the sugar chains among the total complex typeN-glycoside-linked sugar chains which bind to the Fc region contained inthe composition is 20% or more. Examples include cells in which activityof at least one protein described below is decreased or deleted, and thelike:

-   (a) an enzyme protein relating to synthesis of an intracellular    sugar nucleotide, GDP-fucose;-   (b) an enzyme protein relating to the modification of a sugar chain    in which 1-position of fucose is bound to 6-position of    N-acetylglucosamine in the reducing terminal through α-bond in a    complex type N-glycoside-linked sugar chain;-   (c) a protein relating to transport of an intracellular sugar    nucleotide, GDP-fucose, to the Golgi body.

The above host cell is preferably a host cell in which a gene encodingα1,6-fucosyltransferase in the host cell is knocked out (WO02/31140,WO03/85107).

The enzyme protein relating to synthesis of an intracellular sugarnucleotide, GDP-fucose may be any enzyme, so long as it is an enzymerelating to the synthesis of the intracellular sugar nucleotide,GDP-fucose, as a supply source of fucose to a sugar chain. The enzymerelating to synthesis of an intracellular sugar nucleotide, GDP-fucoseincludes an enzyme which has influence on the synthesis of theintracellular sugar nucleotide, GDP-fucose, and the like.

The intracellular sugar nucleotide, GDP-fucose, is supplied by a de novosynthesis pathway or a salvage synthesis pathway. Thus, all enzymesrelating to the synthesis pathways are included in the enzyme relatingto synthesis of an intracellular sugar nucleotide, GDP-fucose.

The enzyme relating to the de novo synthesis pathway of an intracellularsugar nucleotide, GDP-fucose includes GDP-mannose 4,6-dehydratase(hereinafter referred to as “GMD”),GDP-keto-6-deoxymannose-3,5-epimerase, 4,6-reductase (hereinafterreferred to as “Fx”) and the like.

The enzyme relating to the salvage synthesis pathway of an intracellularsugar nucleotide, GDP-fucose includes GDP-beta-L-fucosepyrophosphorylase (hereinafter referred to as “GFPP”), fucokinase andthe like.

As the enzyme which has influence on the synthesis of an intracellularsugar nucleotide, GDP-fucose, an enzyme which has influence on theactivity of the enzyme relating to the synthesis pathway of theintracellular sugar nucleotide, GDP-fucose described above, and anenzyme which has influence on the structure of substances as thesubstrate of the enzyme are also included.

The GDP-mannose 4,6-dehydratase includes:

-   (a) a DNA comprising the nucleotide sequence represented by SEQ ID    NO:18;-   (b) a DNA which hybridizes with the DNA consisting of the nucleotide    sequence represented by SEQ ID NO:18 under stringent conditions and    encodes a protein having GDP-mannose 4,6-dehydratase activity,-   and the like.

The GDP-mannose 4,6-dehydratase includes:

-   (a) a protein comprising the amino acid sequence represented by SEQ    ID NO:19;-   (b) a protein consisting of an amino acid sequence in which one or    more amino acid(s) is/are deleted, substituted, inserted and/or    added in the amino acid sequence represented by SEQ ID NO:19 and    having GDP-mannose 4,6-dehydratase activity;-   (c) a protein consisting of an amino acid sequence which has 80% or    more homology with the amino acid sequence represented by SEQ ID    NO:19 and having GDP-mannose 4,6-dehydratase activity;-   and the like.

The GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase includes:

-   (a) a DNA comprising the nucleotide sequence represented by SEQ ID    NO:20;-   (b) a DNA which hybridizes with the DNA consisting of the nucleotide    sequence represented by SEQ ID NO:20 under stringent conditions and    encodes a protein having GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase    activity;-   and the like.

The GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase includes:

-   (a) a protein comprising the amino acid sequence represented by SEQ    ID NO:21;-   (b) a protein consisting of an amino acid sequence in which one or    more amino acid(s) is/are deleted, substituted, inserted and/or    added in the amino acid sequence represented by SEQ ID NO:21 and    having GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity;-   (c) a protein consisting of an amino acid sequence which has 80% or    more homology with the amino acid sequence represented by SEQ ID    NO:21 and has GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity;-   and the like.

The enzyme protein relating to the modification of a sugar chain inwhich 1-position of fucose is bound to 6-position of N-acetylglucosaminein the reducing terminal through α-bond in a complex typeN-glycoside-linked sugar chain includes any enzyme, so long as it is anenzyme relating to the reaction of binding of 1-position of fucose to6-position of N-acetylglucosamine in the reducing terminal throughα-bond in the complex type N-glycoside-linked sugar chain. The enzymerelating to the reaction of binding of 1-position of fucose to6-position of N-acetylglucosamine in the reducing terminal throughα-bond in the complex type N-glycoside-linked sugar chain includes anenzyme which has influence on the reaction of binding of 1-position offucose to 6-position of N-acetylglucosamine in the reducing terminalthrough α-bond in the complex type N-glycoside-linked sugar chain.Examples include α1,6-fucosyltransferase, α-L-fucosidase and the like.

Also, the enzyme relating to the reaction of binding of 1-position offucose to 6-position of N-acetylglucosamine in the reducing terminalthrough α-bond in the complex type N-glycoside-linked sugar chainincludes an enzyme which has influence on the activity of the enzymerelating to the reaction of binding of 1-position of fucose to6-position of N-acetylglucosamine in the reducing terminal throughα-bond in the complex type N-glycoside-linked sugar chain and an enzymewhich has influence on the structure of substances as the substrate ofthe enzyme.

In the present invention, the α1,6-fucosyltransferase is a proteinencoded by a DNA of the following (a), (b), (c) or (d):

-   (a) a DNA comprising the nucleotide sequence represented by SEQ ID    NO:22;-   (b) a DNA comprising the nucleotide sequence represented by SEQ ID    NO:23;-   (c) a DNA which hybridizes with the DNA consisting of the nucleotide    sequence represented by SEQ ID NO:22 under stringent conditions and    encodes a protein having α1,6-fucosyltransferase activity;-   (d) a DNA which hybridizes with the DNA consisting of the nucleotide    sequence represented by SEQ ID NO:23 under stringent conditions and    encodes a protein having α-1,6-fucosyltransferase activity, or-   (e) a protein comprising the amino acid sequence represented by SEQ    ID NO:24;-   (f) a protein comprising the amino acid sequence represented by SEQ    ID NO:25;-   (g) a protein consisting of an amino acid sequence in which one or    more amino acid(s) is/are deleted, substituted, inserted and/or    added in the amino acid sequence represented by SEQ ID NO:24 and    having α1,6-fucosyltransferase activity;-   (h) a protein consisting of an amino acid sequence in which one or    more amino acid(s) is/are deleted, substituted, inserted and/or    added in the amino acid sequence represented by SEQ ID NO:25 and    having α1,6-fucosyltransferase activity;-   (i) a protein consisting of an amino acid sequence which has 80% or    more homology with the amino acid sequence represented by SEQ ID    NO:24 and having α1,6-fucosyltransferase activity;-   (j) a protein consisting of an amino acid sequence which has 80% or    more homology with the amino acid sequence represented by SEQ ID    NO:25 and having α1,6-fucosyltransferase activity;-   and the like.

The protein relating to transport of an intracellular sugar nucleotide,GDP-fucose, to the Golgi body may be any protein, so long as it is aprotein relating to the transport of the intracellular sugar nucleotide,GDP-fucose, to the Golgi body, or a protein which has an influence onthe reaction for the transport of the intracellular sugar nucleotide,GDP-fucose, to the Golgi body.

The protein relating to the transport of the intracellular sugarnucleotide, GDP-fucose, to the Golgi body includes a GDP-fucosetransporter and the like.

Also, the protein which has an influence on the reaction for thetransport of the intracellular sugar nucleotide, GDP-fucose, to theGolgi body include a protein which has an influence on the activity ofthe above protein relating to the transport of the intracellular sugarnucleotide, GDP-fucose, to the Golgi body or has influence on theexpression thereof.

The DNA encoding the amino acid sequence of the enzyme relating tosynthesis of an intracellular sugar nucleotide, GDP-fucose includes aDNA comprising the nucleotide sequence represented by SEQ ID NO:18 or20; a DNA which hybridizes with the DNA consisting of the nucleotidesequence represented by SEQ ID NO:18 or 20 under stringent conditionsand encodes a protein having activity of the enzyme relating tosynthesis of an intracellular sugar nucleotide, GDP-fucose; and thelike.

The DNA encoding the amino acid sequence of the α1,6-fucosyltransferaseincludes a DNA comprising the nucleotide sequence represented by SEQ IDNO:22 or 23; a DNA which hybridizes with the DNA consisting of thenucleotide sequence represented by SEQ ID NO:22 or 23 under stringentconditions and encodes a protein having α1,6-fucosyltransferaseactivity; and the like.

The method for obtaining a cell in which the above enzyme activity isdecreased or deleted may by any method, so long as it is a method fordecreasing or deleting the objective enzyme activity. Examples include:

-   (a) gene disruption targeting at a gene encoding the enzyme;-   (b) introduction of a dominant-negative mutant of a gene encoding    the enzyme;-   (c) introduction of a mutation into the enzyme;-   (d) suppression of transcription or translation of a gene encoding    the enzyme;-   (e) selection of a cell line resistant to a lectin which recognizes    a sugar chain structure in which 1-position of fucose is bound to    6-position of N-acetylglucosamine in the reducing terminal through    α-bond in a N-glycoside-linked sugar chain;-   and the like.

As the lectin which recognizes a sugar chain structure in which1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing terminal through α-bond in a N-glycoside-linked sugarchain, any lectin capable of recognizing the sugar chain structure canbe used. Specific examples include lentil lectin LCA (lentil agglutininderived from Lens culinaris), pea lectin PSA (pea lectin derived fromPisum sativum), broad bean lectin VFA (agglutinin derived from Viciafaba), Aleuria aurantia lectin AAL (lectin derived from Aleuriaaurantia) and the like.

The “cell resistant to a lectin” refers to a cell in which growth is notinhibited by the presence of a lectin at an effective concentration. The“effective concentration” is a concentration higher than theconcentration that does not allow the normal growth of a cell prior tothe genome modification (hereinafter referred to also as parent cellline), preferably equal to the concentration that does not allow thenormal growth of a cell prior to the genome modification, morepreferably 2 to 5 times, further preferably 10 times, most preferably 20or more times the concentration that does not allow the normal growth ofa cell prior to the modification of the genomic gene.

The effective concentration of lectin that does not inhibit growth maybe appropriately determined according to each cell line. It is usually10 μg/ml to 10 mg/ml, preferably 0.5 mg/ml to 2.0 mg/ml.

Processes for producing the recombinant antibody composition of thepresent invention are explained below in detail.

1. Process for Producing Recombinant Antibody Composition

The recombinant antibody composition of the present invention can beobtained, for example, by expressing it in a host cell using the methodsdescribed in Molecular Cloning, Second Edition; Current Protocols inMolecular Biology; Antibodies, A Laboratory manual, Cold Spring HarborLaboratory (1988) (hereinafter referred to as Antibodies); MonoclonalAntibodies: principles and practice, Third Edition, Acad. Press (1993)(hereinafter referred to as Monoclonal Antibodies); AntibodyEngineering, A Practical Approach, IRL Press at Oxford University Press,1996 (hereinafter referred to as Antibody Engineering); and the like,for example, in the following manner.

(1) Construction of a Vector for Expression of the Recombinant AntibodyComposition of the Present Invention

A vector for expression of the recombinant antibody composition of thepresent invention is an expression vector for animal cell into whichgenes encoding H chain and L chain constant regions of an antibodymolecule contained in the recombinant antibody composition of thepresent invention are introduced. The vector for expression of therecombinant antibody composition can be constructed by cloning each ofthe genes encoding H chain and L chain constant regions of an antibodymolecule contained in the recombinant antibody composition into a vectorfor expression of animal cell.

The gene encoding the CH region of an antibody molecule contained in therecombinant antibody composition of the present invention can beproduced by cloning genes encoding constant regions of IgG1 and IgG3antibodies and then ligating gene fragments encoding respective domains.Also, the total DNA can be synthesized by using synthetic DNAs andsynthesis using PCR can also be carried out (Molecular Cloning, SecondEdition). Furthermore, it can be produced by combining these techniques.

The expression vector for animal cell may by any vector, so long as theabove gene encoding the constant region of an antibody molecule can beintroduced and expressed. Examples include pKANTEX93 [Mol. Immunol., 37,1035 (2000)], pAGE107 [Cytotechnology, 3, 133 (1990), pAGE103 [J.Biochem., 101, 1307 (1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR [Proc.Natl. Acad. Sci. U.S.A., 78, 1527 (1981)], pSG1βd2-4 [Cytotechnology, 4,173 (1990)] and the like. The promoter and enhancer used for theexpression vector for animal cell include SV40 early promoter andenhancer [J. Biochem., 101, 1307 (1987)], LTR of Moloney mouse leukemiavirus [Biochem. Biophys. Res. Commun., 149, 960 (1987)], immunoglobulinH chain promoter [Cell, 41, 479 (1985)] and ehnancer [Cell, 33, 717(1983)] and the like.

The vector for expression of the recombinant antibody composition of thepresent invention may be either of a type in which genes encoding the Hchain and L chain of antibody exist on separate vectors or of a type inwhich both genes exist on the same vector (tandem type). In respect ofeasiness of construction of a vector for expression of the recombinantantibody composition of the present invention, easiness of introductioninto animal cells, and balance between the expression amounts of the Hand L chains of antibody in animal cells, a tandem type of the vectorfor expression of humanized antibody is more preferred (J. Immunol.Methods, 167, 271 (1994)).

The constructed vector for expression of the recombinant antibodycomposition of the present invention can be used for expression of ahuman chimeric antibody and a humanized antibody in animal cells.

(2) Obtaining of cDNA Encoding V Region of Non-Human Animal Antibody

cDNAs encoding VH and VL of a non-human animal antibody such as a mouseantibody can be obtained in the following manner.

A cDNA is synthesized by using as a probe mRNA extracted from ahybridoma cell which produces any antibody. The synthesized cDNA iscloned into a vector such as a phage or a plasmid to obtain a cDNAlibrary. Each of a recombinant phage or recombinant plasmid comprising acDNA encoding the H chain V region and a recombinant phage orrecombinant plasmid comprising a cDNA encoding the L chain V region isisolated from the library by using cDNA encoding C region or V region ofa known mouse antibody as the probe. Full length nucleotide sequences ofVH and VL of the mouse antibody of interest on the recombinant phage orrecombinant plasmid are determined, and full length amino acid sequencesof VH and VL are deduced from the nucleotide sequences.

Hybridoma cells producing any non-human animal-derived antibody can beobtained by immunizing a non-human animal with an antigen bound to theantibody, preparing hybridomas from antibody-producing cells of theimmunized animal and myeloma cells according to a known method[Molecular Cloning, Second Edition; Current Protocols in MolecularBiology; Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory(1988) (hereinafter referred to as Antibodies); Monoclonal Antibodies:principles and practice, Third Edition, Acad. Press (1993) (hereinafterreferred to as Monoclonal Antibodies), Antibody Engineering, A PracticalApproach, IRL Press at Oxford University Press (1996) (hereinafterreferred to as Antibody Engineering)], selecting cloned hybridomas,culturing the selected hybridomas and purifying cells from the culturesupernatant.

As the non-human animal, any animal can be used so long as hybridomacells can be prepared from the animal. Suitable animals include mouse,rat, hamster and rabbit.

The methods for preparing total RNA from a hybridoma cell include theguanidine thiocyanate-cesium trifluoroacetate method [Methods inEnzymol., 154, 3 (1987)], and the methods for preparing mRNA from thetotal RNA include the oligo (dT) immobilized cellulose column method[Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Lab. Press(1989)]. Examples of the kits for preparing mRNA from a hybridoma cellinclude Fast Track mRNA Isolation Kit (manufactured by Invitrogen) andQuick Prep mRNA Purification Kit (manufactured by Pharmacia).

The methods for synthesizing the cDNA and preparing the cDNA libraryinclude conventional methods [Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Lab. Press (1989), Current Protocols in MolecularBiology, Supplement 1-34], or methods using commercially available kitssuch as SuperScript™ Plasmid System for cDNA Synthesis and PlasmidCloning (manufactured by GIBCO BRL) and ZAP-cDNA Synthesis Kit(manufactured by Stratagene).

In preparing the cDNA library, the vector for integrating the cDNAsynthesized using the mRNA extracted from a hybridoma cell as a templatemay be any vector so long as the cDNA can be integrated. Examples ofsuitable vectors include ZAP Express [Strategies, 5, 58 (1992)],pBluescript II SK(+) [Nucleic Acids Research, 17, 9494 (1989)], λZAP II(manufactured by STRATAGENE), λgt10, λgt11 [DNA Cloning: A PracticalApproach, I, 49 (1985)], Lambda BlueMid (manufactured by Clontech),λExCell, pT7T3 18U (manufactured by Pharmacia), pcD2 [Mol. Cell. Biol.,3, 280 (1983)], pUC18 [Gene, 33, 103 (1985)] and the like.

As Escherichia coli for introducing the cDNA library constructed with aphage or plasmid vector, any Escherichia coli can be used so long as thecDNA library can be introduced, expressed and maintained. Examples ofsuitable Escherichia coli include XL1-Blue MRF′ [Strategies, 5, 81(1992)], C600 [Genetics, 39, 440 (1954)], Y1088, Y1090 [Science, 222,778 (1983)], NM522 [J. Mol. Biol., 166, 1 (1983)], K802 [J. Mol. Biol.,16, 118 (1966)], JM105 [Gene, 38, 275 (1985)] and the like.

The methods for selecting the cDNA clones encoding VH and VL of anon-human animal-derived antibody from the cDNA library include colonyhybridization or plaque hybridization [Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press (1989)] using an isotope- orfluorescence-labeled probe. It is also possible to prepare the cDNAsencoding VH and VL by preparing primers and carrying out PCR [MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press(1989), Current Protocols in Molecular Biology, Supplement 1-34] usingthe cDNA or cDNA library as a template.

The nucleotide sequences of the cDNAs selected by the above methods canbe determined by cleaving the cDNAs with appropriate restrictionenzymes, cloning the fragments into a plasmid such as pBluescript SK(−)(manufactured by STRATAGENE), and then analyzing the sequences bygenerally employed nucleotide sequence analyzing methods such as thedideoxy method of Sanger, et al. [Proc. Natl. Acad. Sci. USA, 74, 5463(1977)] or by use of nucleotide sequence analyzers such as ABI PRISM 377DNA Sequencer (manufactured by Applied Biosystems).

The full length of amino acid sequences of VH and VL are deduced fromthe determined nucleotide sequences and compared with the full length ofamino acid sequences of VH and VL of a known antibody [Sequences ofProteins of Immunological Interest, US Dept. Health and Human Services(1991)], whereby it can be confirmed that the obtained cDNAs encodeamino acid sequences which completely comprise VH and VL of the antibodyincluding secretory signal sequences.

Further, when the amino acid sequence of an antibody variable region orthe nucleotide sequence of DNA encoding the variable region is alreadyknown, the DNA can be obtained by the following methods.

When the amino acid sequence is known, the DNA can be obtained bydesigning a DNA sequence encoding the variable region taking intoconsideration the frequency of codon usage [Sequences of Proteins ofImmunological Interest, US Dept. Health and Human Services (1991)],synthesizing several synthetic DNAs constituting approximately100-nucleotides based on the designed DNA sequence, and carrying out PCRusing the synthetic DNAs. When the nucleotide sequence is known, the DNAcan be obtained by synthesizing several synthetic DNAs constitutingapproximately 100-nucleotides based on the nucleotide sequenceinformation and carrying out PCR using the synthetic DNAs.

(3) Analysis of the Amino Acid Sequence of the V Region of an Antibodyfrom a Non-Human Animal

By comparing the full length of amino acid sequences of VH and VL of theantibody including secretory signal sequences with the amino acidsequences of VH and VL of a known antibody [Sequences of Proteins ofImmunological Interest, US Dept. Health and Human Services (1991)], itis possible to deduce the length of the secretory signal sequences andthe N-terminal amino acid sequences and further to know the subgroup towhich the antibody belongs. In addition, the amino acid sequences ofCDRs of VH and VL can be deduced in a similar manner.

(4) Construction of a Human Chimeric Antibody Expression Vector

A human chimeric antibody expression vector can be constructed byinserting the cDNAs encoding VH and VL of an antibody of a non-humananimal into sites upstream of the genes encoding CH and CL of a humanantibody in the vector for expression of recombinant antibodycomposition described in the above 1 (1). For example, a human chimericantibody expression vector can be constructed by ligating the cDNAsencoding VH and VL of an antibody of a non-human animal respectively tosynthetic DNAs comprising the 3′-terminal nucleotide sequences of VH andVL of an antibody of a non-human animal and the 5′-terminal nucleotidesequences of CH and CL of a human antibody and also having recognitionsequences for appropriate restriction enzymes at both ends, andinserting them into sites upstream of the genes encoding CH and CL of ahuman antibody in the vector for recombinant antibody compositiondescribed in the above 1 (1) so as to express them in an appropriateform.

(5) Construction of cDNA Encoding V Region of a Humanized Antibody

cDNAs encoding VH and VL of a humanized antibody can be constructed inthe following manner. First, amino acid sequences of FRs of VH and VL ofa human antibody for grafting CDRs of VH and VL of a non-humananimal-derived antibody are selected. The amino acid sequences of FRs ofVH and VL of a human antibody may be any of those from human antibodies.Suitable sequences include the amino acid sequences of FRs of VHs andVLs of human antibodies registered at databases such as Protein DataBank, and the amino acid sequences common to subgroups of FRs of VHs andVLs of human antibodies [Sequences of Proteins of ImmunologicalInterest, US Dept. Health and Human Services (1991)]. In order toprepare a humanized antibody having a sufficient activity, it ispreferred to select amino acid sequences having a homology of as high aspossible (at least 60% or more) with the amino acid sequences of FRs ofVH and VL of the desired non-human animal-derived antibody.

Next, the amino acid sequences of CDRs of VH and VL of the desirednon-human animal-derived antibody are grafted to the selected amino acidsequences of FRs of VH and VL of a human antibody to design amino acidsequences of VH and VL of a humanized antibody. The designed amino acidsequences are converted into DNA sequences taking into consideration thefrequency of codon usage in the nucleotide sequences of antibody genes[Sequences of Proteins of Immunological Interest, US Dept. Health andHuman Services (1991)], and DNA sequences encoding the amino acidsequences of VH and VL of the humanized antibody are designed. Severalsynthetic DNAs constituting approximately 100-nucleotides aresynthesized based on the designed DNA sequences, and PCR is carried outusing the synthetic DNAs. It is preferred to design 4 to 6 syntheticDNAs for each of the H chain and the L chain in view of the reactionefficiency of PCR and the lengths of DNAs that can be synthesized.

Cloning into the vector for expression of the recombinant antibodycomposition of the present invention constructed in the above 1 (1) canbe easily carried out by introducing recognition sequences forappropriate restriction enzymes to the 5′-terminals of synthetic DNAspresent on both ends. After the PCR, the amplification products arecloned into a plasmid such as pBluescript SK(−) (manufactured bySTRATAGENE) and the nucleotide sequences are determined by the methoddescribed in the above 1 (2) to obtain a plasmid carrying DNA sequencesencoding the amino acid sequences of VH and VL of the desired humanizedantibody.

(6) Modification of the Amino Acid Sequence of V Region of a HumanizedAntibody

It is known that a humanized antibody prepared merely by grafting CDRsof VH and VL of a non-human animal-derived antibody to FRs of VH and VLof a human antibody has a lower antigen-binding activity compared withthe original non-human animal-derived antibody [BIO/TECHNOLOGY, 9, 266(1991)]. This is probably because in VH and VL of the original non-humananimal-derived antibody, not only CDRs but also some of the amino acidresidues in FRs are involved directly or indirectly in theantigen-binding activity, and such amino acid residues are replaced byamino acid residues of FRs of VH and VL of the human antibody by CDRgrafting. In order to solve this problem, attempts have been made in thepreparation of a humanized antibody to raise the lowered antigen-bindingactivity by identifying the amino acid residues in the amino acidsequences of FRs of VH and VL of a human antibody which are directlyrelating to the binding to an antigen or which are indirectly relatingto it through interaction with amino acid residues in CDRs ormaintenance of the three-dimensional structure of antibody, andmodifying such amino acid residues to those derived from the originalnon-human animal-derived antibody [BIO/TECHNOLOGY, 9, 266 (1991)].

In the preparation of a humanized antibody, it is most important toefficiently identify the amino acid residues in FR which are relating tothe antigen-binding activity. For the efficient identification,construction and analyses of the three-dimensional structures ofantibodies have been carried out by X ray crystallography [J. Mol.Biol., 112, 535 (1977)], computer modeling [Protein Engineering, 7, 1501(1994)], etc. Although these studies on the three-dimensional structuresof antibodies have provided much information useful for the preparationof humanized antibodies, there is no established method for preparing ahumanized antibody that is adaptable to any type of antibody. That is,at present, it is still necessary to make trial-and-error approaches,e.g., preparation of several modifications for each antibody andexamination of each modification for the relationship with theantigen-binding activity.

Modification of the amino acid residues in FRs of VH and VL of a humanantibody can be achieved by PCR as described in the above 1 (5) usingsynthetic DNAs for modification. The nucleotide sequence of the PCRamplification product is determined by the method described in the above1 (2) to confirm that the desired modification has been achieved.

(7) Construction of a Humanized Antibody Expression Vector

A humanized antibody expression vector can be constructed by insertingthe cDNAs encoding VH and VL of the humanized antibody constructed inthe above 1 (5) and (6) into sites upstream of the genes encoding CH andCL of a human antibody in the vector for expression of the recombinantantibody composition of the present invention described in the above 1(1). For example, a humanized antibody expression vector can beconstructed by introducing recognition sequences for appropriaterestriction enzymes to the 5′-terminals of synthetic DNAs present onboth ends among the synthetic DNAs used for constructing VH and VL ofthe humanized antibody in the above 1 (5) and (6), and inserting theminto sites upstream of the genes encoding CH and CL of a human antibodyin the vector for expression of the recombinant antibody of the presentinvention described in the above 1 (1) so as to express them in anappropriate form.

(8) Stable Production of a Humanized Antibody

Transformants capable of stably producing a human chimeric antibody anda humanized antibody can be obtained by introducing the human chimericantibody or humanized antibody expression vectors described in the above1 (4) and (7) into appropriate animal cells.

Introduction of the humanized antibody expression vector into an animalcell can be carried out by electroporation [Japanese PublishedUnexamined Patent Application No. 257891/90; Cytotechnology, 3, 133(1990)], etc.

As the animal cell for introducing the human chimeric antibody orhumanized antibody expression vector, any animal cell capable ofproducing a human chimeric antibody or a humanized antibody can be used.

Examples of the animal cells include mouse myeloma cell lines NS0 andSP2/0, Chinese hamster ovary cells CHO/dhfr− and CHO/DG44, rat myelomacell lines YB2/0 and IR983F, Syrian hamster kidney-derived BHK cell, andhuman myeloma cell line Namalwa. Chinese hamster ovary cell CHO/DG44 andrat myeloma cell line YB2/0 are preferred.

After the introduction of the human chimeric antibody or humanizedantibody expression vector, the transformant capable of stably producingthe human chimeric antibody or the humanized antibody can be selectedusing a medium for animal cell culture containing an agent such as G418sulfate (hereinafter referred to as G418; manufactured by SIGMA)according to the method described in Japanese Published UnexaminedPatent Application No. 257891/90. Examples of the media for animal cellculture include RPMI1640 medium (manufactured by Nissui PharmaceuticalCo., Ltd.), GIT medium (manufactured by Nihon Pharmaceutical Co., Ltd.),EX-CELL 302 medium (manufactured by JRH), IMDM medium (manufactured byGIBCO BRL), Hybridoma-SFM medium (manufactured by GIBCO BRL), and mediaprepared by adding various additives such as fetal calf serum(hereinafter referred to as FCS) to these media. By culturing theobtained transformant in the medium, the human chimeric antibody or thehumanized antibody can be formed and accumulated in the culturesupernatant. The amount and the antigen-binding activity of the humanchimeric antibody or the humanized antibody produced in the culturesupernatant can be measured by enzyme-linked immunosorbent assay[hereinafter referred to as ELISA; Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, Chapter 14 (1998); Monoclonal Antibodies:Principles and Practice, Academic Press Limited (1996)] or the like. Theamount of the human chimeric antibody or the humanized antibody to beproduced by the transformant can be increased by utilizing a DHFR geneamplification system or the like according to the method described inJapanese Published Unexamined Patent Application No. 257891/90.

The human chimeric antibody or the humanized antibody can be purifiedfrom the culture supernatant of the transformant using a protein Acolumn [Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Chapter 8 (1988); Monoclonal Antibodies: Principles and Practice,Academic Press Limited (1996)]. In addition, purification methodsgenerally employed for the purification of proteins can also be used.For example, the purification can be carried out by combinations of gelfiltration, ion exchange chromatography, ultrafiltration and the like.The molecular weight of the H chain, L chain or whole antibody moleculeof the purified human chimeric antibody or humanized antibody can bemeasured by SDS-denatured polyacrylamide gel electrophoresis[hereinafter referred to as SDS-PAGE; Nature, 227, 680 (1970)], Westernblotting [Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, Chapter 12 (1988); Monoclonal Antibodies: Principles andPractice, Academic Press Limited (1996)], etc.

Shown above is the method for producing the antibody composition usingan animal cell as the host. The antibody composition can also beproduced using yeast, an insect cell, a plant cell, an animal individualor a plant individual by similar methods.

Accordingly, when the host cell is capable of expressing an antibodymolecule, the antibody composition of the present invention can beproduced by introducing a gene encoding an antibody into the host cellwhich expresses an antibody molecule, culturing the cell, and purifyingthe desired antibody composition from the culture.

When yeast is used as the host cell, YEP13 (ATCC 37115), YEp24 (ATCC37051), YCp50 (ATCC 37419), etc. can be used as the expression vector.

As the promoter, any promoters capable of expressing in yeast strainscan be used. Suitable promoters include promoters of genes of theglycolytic pathway such as hexosekinase, PHO5 promoter, PGK promoter,GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shockprotein promoter, MFα1 promoter and CUP 1 promoter.

Examples of suitable host cells are microorganisms belonging to thegenera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporonand Schwanniomyces, and specifically, Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans,Schwanniomyces alluvius and the like.

Introduction of the recombinant vector can be carried out by any of themethods for introducing DNA into yeast, for example, electroporation[Methods Enzymol., 194, 182 (1990)], the spheroplast method [Proc. Natl.Acad. Sci. USA, 84, 1929 (1978)], the lithium acetate method [J.Bacteriology, 153, 163 (1983)] and the method described in Proc. Natl.Acad. Sci. USA, 75, 1929 (1978).

When an animal cell is used as the host cell, pcDNAI, pcDM8(commercially available from Funakoshi Co., Ltd.), pAGE107 [JapanesePublished Unexamined Patent Application No. 22979/91; Cytotechnology, 3,133 (1990)], pAS3-3 (Japanese Published Unexamined Patent ApplicationNo. 227075/90), pCDM8 [Nature, 329, 840 (1987)], pcDNAI/Amp(manufactured by Invitrogen Corp.), pREP4 (manufactured by InvitrogenCorp.), pAGE103 [J. Biochemistry, 101, 1307 (1987)], pAGE210, etc. canbe used as the expression vector.

As the promoter, any promoters capable of expressing in animal cells canbe used. Suitable promoters include the promoter of IE (immediate early)gene of cytomegalovirus (CMV), SV40 early promoter, the promoter of aretrovirus, metallothionein promoter, heat shock promoter, SRα promoter,etc. The enhancer of IE gene of human CMV may be used in combinationwith the promoter.

Examples of suitable host cells are human-derived Namalwa cells,monkey-derived COS cells, Chinese hamster-derived CHO cells, HBT5637(Japanese Published Unexamined Patent Application No. 299/88), ratmyeloma cells, mouse myeloma cells, cells derived from Syrian hamsterkidney, embryonic stem cells, fertilized egg cells and the like.

When an insect cell is used as the host cell, the protein can beexpressed by the methods described in Current Protocols in MolecularBiology; Baculovirus Expression Vectors, A Laboratory Manual, W. H.Freeman and Company, New York (1992); Bio/Technology, 6, 47 (1988), etc.

That is, the expression vector and a baculovirus are cotransfected intoinsect cells to obtain a recombinant virus in the culture supernatant ofthe insect cells, and then insect cells are infected with therecombinant virus, whereby the protein can be expressed.

The gene introducing vectors useful in this method include pVL1392,pVL1393, pBlueBacIII (products of Invitrogen Corp.) and the like.

An example of the baculovirus is Autographa californica nuclearpolyhedrosis virus, which is a virus infecting insects belonging to thefamily Barathra.

Examples of the insect cells are Spodoptera frugiperda ovarian cells Sf9and Sf21 [Current Protocols in Molecular Biology; Baculovirus ExpressionVectors, A Laboratory Manual, W.H. Freeman and Company, New York (1992)]and Trichoplusia ni ovarian cell High 5 (manufactured by InvitrogenCorp.).

Cotransfection of the above expression vector and the above baculovirusinto insect cells for the preparation of the recombinant virus can becarried out by the calcium phosphate method (Japanese PublishedUnexamined Patent Application No. 227075/90), lipofection [Proc. Natl.Acad. Sci. USA, 84, 7413 (1987)], etc.

When a plant cell is used as the host cell, Ti plasmid, tobacco mosaicvirus vector, etc. can be used as the expression vector.

As the promoter, any promoters capable of expressing in plant cells canbe used. Suitable promoters include 35S promoter of cauliflower mosaicvirus (CaMV), rice actin 1 promoter, etc.

Examples of suitable host cells are cells of plants such as tobacco,potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat and barley.

Introduction of the recombinant vector can be carried out by any of themethods for introducing DNA into plant cells, for example, the methodusing Agrobacterium (Japanese Published Unexamined Patent ApplicationNos. 140885/84 and 70080/85, WO94/00977), electroporation (JapanesePublished Unexamined Patent Application No. 251887/85) and the methodusing particle gun (gene gun) (Japanese Patent Nos. 2606856 and2517813).

Introduction of the recombinant vector can be carried out by any of themethods for introducing DNA into animal cells, for example,electroporation [Cytotechnology, 3, 133 (1990)], the calcium phosphatemethod (Japanese Published Unexamined Patent Application No. 227075/90),lipofection [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], the injectionmethod (Manipulating the Mouse Embryo, A Laboratory Manual), the methodusing particle gun (gene gun) (Japanese Patent Nos. 2606856 and2517813), the DEAE-dextran method [Biomanual Series 4—Methods of GeneTransfer, Expression and Analysis (Yodosha), edited by Takashi Yokotaand Kenichi Arai (1994)] and the virus vector method (Manipulating theMouse Embryo, A Laboratory Manual).

Expression of the gene encoding the antibody can be carried out not onlyby direct expression but also by secretory production, expression of afusion protein of the Fc region and another protein, etc. according tothe methods described in Molecular Cloning, Second Edition.

The antibody composition can be produced by culturing the transformantobtained as above in a medium, allowing the antibody molecules to formand accumulate in the culture, and recovering them from the culture.Culturing of the transformant in a medium can be carried out byconventional methods for culturing the host cell.

For the culturing of the transformant obtained by using a eucaryote suchas yeast as the host, any of natural media and synthetic media can beused insofar as it is a medium suitable for efficient culturing of thetransformant which contains carbon sources, nitrogen sources, inorganicsalts, etc. which can be assimilated by the host used.

As the carbon sources, any carbon sources that can be assimilated by thehost can be used. Examples of suitable carbon sources includecarbohydrates such as glucose, fructose, sucrose, molasses containingthem, starch and starch hydrolyzate; organic acids such as acetic acidand propionic acid; and alcohols such as ethanol and propanol.

As the nitrogen sources, ammonia, ammonium salts of organic or inorganicacids such as ammonium chloride, ammonium sulfate, ammonium acetate andammonium phosphate, and other nitrogen-containing compounds can be usedas well as peptone, meat extract, yeast extract, corn steep liquor,casein hydrolyzate, soybean cake, soybean cake hydrolyzate, and variousfermented microbial cells and digested products thereof.

Examples of the inorganic salts include potassium dihydrogenphosphate,dipotassium hydrogenphosphate, magnesium phosphate, magnesium sulfate,sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate,calcium carbonate and the like.

Culturing is usually carried out under aerobic conditions, for example,by shaking culture or submerged spinner culture under aeration. Theculturing temperature is preferably 15 to 40° C., and the culturingperiod is usually 16 hours to 7 days. The pH is maintained at 3.0 to 9.during the culturing. The pH adjustment is carried out by using anorganic or inorganic acid, an alkali solution, urea, calcium carbonate,ammonia, etc.

If necessary, antibiotics such as ampicillin and tetracycline may beadded to the medium during the culturing.

When a microorganism transformed with a recombinant vector using aninducible promoter is cultured, an inducer may be added to the medium,if necessary. For example, in the case of a microorganism transformedwith a recombinant vector using lac promoter,isopropyl-β-D-thiogalactopyranoside or the like may be added to themedium; and in the case of a microorganism transformed with arecombinant vector using trp promoter, indoleacrylic acid or the likemay be added.

For the culturing of the transformant obtained by using an animal cellas the host, generally employed media such as RPMI1640 medium [TheJournal of the American Medical Association, 199, 519 (1967)], Eagle'sMEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM medium[Virology, 8, 396 (1959)], 199 medium [Proceeding of the Society for theBiological Medicine, 73, 1 (1950)] and Whitten's medium [DevelopmentalEngineering Experimentation Manual—Preparation of Transgenic Mice(Kodansha), edited by Motoya Katsuki (1987)], media prepared by addingfetal calf serum or the like to these media, etc. can be used as themedium.

Culturing is usually carried out under conditions of pH 6.0 to 8.0 at 30to 40° C. for 1 to 7 days in the presence of 5% CO₂.

If necessary, antibiotics such as kanamycin and penicillin may be addedto the medium during the culturing.

For the culturing of the transformant obtained by using an insect cellas the host, generally employed media such as TNM-FH medium(manufactured by Pharmingen, Inc.), Sf-900 II SFM medium (manufacturedby Life Technologies, Inc.), ExCell 400 and ExCell 405 (manufactured byJRH Biosciences, Inc.) and Grace's Insect Medium [Nature, 195, 788(1962)] can be used as the medium.

Culturing is usually carried out under conditions of pH 6.0 to 7.0 at 25to 30° C. for 1 to 5 days.

If necessary, antibiotics such as gentamicin may be added to the mediumduring the culturing.

The transformant obtained by using a plant cell as the host may becultured in the form of cells as such or after differentiation intoplant cells or plant organs. For the culturing of such transformant,generally employed media such as Murashige-Skoog (MS) medium and Whitemedium, media prepared by adding phytohormones such as auxin andcytokinin to these media, etc. can be used as the medium.

Culturing is usually carried out under conditions of pH 5.0 to 9.0 at 20to 40° C. for 3 to 60 days.

If necessary, antibiotics such as kanamycin and hygromycin may be addedto the medium during the culturing.

As described above, the antibody composition can be produced byculturing, according to a conventional culturing method, thetransformant derived from an animal cell or a plant cell and carrying anexpression vector into which DNA encoding the antibody molecule has beenintegrated, allowing the antibody composition to form and accumulate,and recovering the antibody composition from the culture.

Expression of the gene encoding the antibody can be carried out not onlyby direct expression but also by secretory production, fusion proteinexpression, etc. according to the methods described in MolecularCloning, Second Edition.

The antibody composition may be produced by intracellular expression inhost cells, may be produced by extracellular secretion from host cellsor may be produced on outer membranes of host cells. A desirableproduction method can be adopted by changing the kind of the host cellsused or the structure of the antibody molecule to be produced.

When the antibody composition is produced in host cells or on outermembranes of host cells, it is possible to force the antibodycomposition to be secreted outside the host cells by applying the methodof Paulson, et al. [J. Biol. Chem., 264, 17619 (1989)], the method ofLowe, et al. [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989); GenesDevelop., 4, 1288 (1990)], or the methods described in JapanesePublished Unexamined Patent Application No. 336963/93, WO94/23021, etc.

That is, it is possible to force the desired antibody molecule to besecreted outside the host cells by inserting DNA encoding the antibodymolecule and DNA encoding a signal peptide suitable for the expressionof the antibody molecule into an expression vector, introducing theexpression vector into the host cells, and then expressing the antibodymolecule by use of recombinant DNA techniques.

It is also possible to increase the amount of the antibody compositionto be produced by utilizing a gene amplification system using adihydrofolate reductase gene or the like according to the methoddescribed in Japanese Published Unexamined Patent Application No.227075/90.

Further, the antibody composition can be produced using an animalindividual into which a gene is introduced (non-human transgenic animal)or a plant individual into which a gene is introduced (transgenic plant)constructed by redifferentiating the animal or plant cells into whichgenes are introduced.

When the transformant is an animal individual or plant individual, theantibody composition can be produced by rearing or cultivating theanimal or plant in a usual manner, allowing the antibody composition toform and accumulate therein, and collecting the antibody compositionfrom the animal individual or plant individual.

Production of the antibody composition using an animal individual can becarried out, for example, by producing the desired antibody compositionin an animal constructed by introducing the gene according to knownmethods [American Journal of Clinical Nutrition, 63, 639S (1996);American Journal of Clinical Nutrition, 63, 627S (1996); Bio/Technology,9, 830 (1991)].

In the case of an animal individual, the antibody composition can beproduced, for example, by raising a non-human transgenic animal intowhich DNA encoding the antibody molecule is introduced, allowing theantibody composition to form and accumulate in the animal, andcollecting the antibody composition from the animal. The places wherethe antibody composition is formed and accumulated include milk(Japanese Published Unexamined Patent Application No. 309192/88), egg orthe like of the animal. As the promoter in this process, any promoterscapable of expressing in an animal can be used. Preferred promotersinclude mammary gland cell-specific promoters such as a casein promoter,β casein promoter, β lactoglobulin promoter and whey acidic proteinpromoter.

Production of the antibody composition using a plant individual can becarried out, for example, by cultivating a transgenic plant into whichDNA encoding the antibody molecule is introduced according to knownmethods [Soshiki Baiyo (Tissue Culture), 20 (1994); Soshiki Baiyo(Tissue Culture), 21 (1995); Trends in Biotechnology, 15, 45 (1997)],allowing the antibody composition to form and accumulate in the plant,and collecting the antibody composition from the plant.

When the antibody composition produced by the transformant into whichthe gene encoding the antibody molecule is introduced is expressed in asoluble form in cells, the cells are recovered by centrifugation afterthe completion of culturing and suspended in an aqueous buffer, followedby disruption using a sonicator, French press, Manton Gaulin™homogenizer, Dynomill™ or the like to obtain a cell-free extract. Apurified preparation of the antibody composition can be obtained bycentrifuging the cell-free extract to obtain the supernatant and thensubjecting the supernatant to ordinary means for isolating and purifyingenzymes, e.g., extraction with a solvent, salting-out with ammoniumsulfate, etc., desalting, precipitation with an organic solvent, anionexchange chromatography using resins such as diethylaminoethyl(DEAE)-Sepharose and DIAION™ HPA-75 (manufactured by Mitsubishi ChemicalCorporation), cation exchange chromatography using resins such asS-Sepharose FF (manufactured by Pharmacia), hydrophobic chromatographyusing resins such as butyl Sepharose and phenyl Sepharose, gelfiltration using a molecular sieve, affinity chromatography,chromatofocusing, and electrophoresis such as isoelectric focusing,alone or in combination.

When the antibody composition is expressed as an insoluble body incells, the cells are similarly recovered and disrupted, followed bycentrifugation to recover the insoluble body of the antibody compositionas a precipitate fraction. The recovered insoluble body of the antibodycomposition is solubilized with a protein-denaturing agent. Thesolubilized antibody solution is diluted or dialyzed, whereby theantibody composition is renatured to have normal three-dimensionalstructure. Then, a purified preparation of the antibody composition canbe obtained by the same isolation and purification steps as describedabove.

When the antibody composition is extracellularly secreted, the antibodycomposition or its derivative can be recovered in the culturesupernatant. That is, the culture is treated by the same means as above,e.g., centrifugation, to obtain the culture supernatant. A purifiedpreparation of the antibody composition can be obtained from the culturesupernatant by using the same isolation and purification methods asdescribed above.

2. Preparation of Recombinant Antibody Composition-Producing Cell of thePresent Invention

The cell producing the recombinant antibody composition having high ADCCactivity as well as high CDC activity among the recombinant antibodycompositions of the present invention can be produced by preparing ahost cell used for the production of the recombinant antibodycomposition of the present invention by the following techniques andthen introducing the human chimeric antibody or humanized antibodyexpression vector described in the above 1 (4) and (7) into the hostcell.

Specifically, a cell in which an enzyme relating to the modification ofthe N-glycoside-linked sugar chain bound to the Fc region of an antibodymolecule, that is, an enzyme relating to the synthesis of anintracellular sugar nucleotide, GDP-fucose and/or an enzyme relating tothe modification of a sugar chain in which 1-position of fucose is boundto 6-position of N-acetylglucosamine in the reducing terminal throughα-bond in the complex type N-glycoside-linked sugar chain is inactivatedis selected, or a cell obtained by various artificial techniquesdescribed below can be used as a host cell. The details are describedbelow.

(1) Gene Disruption Technique Targeting at a Gene Encoding an Enzyme

The host cell used for the production of the cell producing the antibodyhaving high ADCC activity (hereinafter referred to as high ADCC activityantibody) can be prepared by a gene disruption technique targeting agene encoding an enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose or an enzyme relating to the modificationof a sugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain. Examples of the enzymes relating tothe synthesis of an intracellular sugar nucleotide, GDP-fucose includeGDP-mannose 4,6-dehydratase (hereinafter referred to as GMD) andGDP-4-keto-6-deoxy-D-mannose-3,5-epimerase (hereinafter referred to asFx).

Examples of the enzymes relating to the modification of a sugar chain inwhich 1-position of fucose is bound to 6-position of N-acetylglucosaminein the reducing terminal through α-bond in a complex typeN-glycoside-linked sugar chain include α1,6-fucosyltransferase,α-L-fucosidase, and the like. The gene as used herein includes DNA andRNA.

The method of gene disruption may be any method capable of disruptingthe gene encoding the enzyme. Useful methods include the antisensemethod, the ribozyme method, the homologous recombination method, theRNA-DNA oligonucleotide method (hereinafter referred to as the RDOmethod), the RNA interference method (hereinafter referred to as theRNAi method), the method using a retrovirus and the method using atransposon, and the like. These methods are specifically describedbelow.

(a) Preparation of the Host Cell for the Production of the High ADCCActivity Antibody-Producing Cell by the Antisense Method or the RibozymeMethod

The host cell used for the production of the high ADCC activityantibody-producing cell can be prepared by the antisense method or theribozyme method described in Cell Technology, 12, 239 (1993);BIO/TECHNOLOGY, 17, 1097 (1999); Hum. Mol. Genet., 5, 1083 (1995); CellTechnology, 13, 255 (1994); Proc. Natl. Acad. Sci. U.S.A., 96, 1886(1999); and the like targeting at a gene encoding an enzyme relating tothe synthesis of an intracellular sugar nucleotide, GDP-fucose or anenzyme relating to the modification of a sugar chain in which 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingterminal through α-bond in a complex type N-glycoside-linked sugarchain, for example, in the following manner.

A cDNA or a genomic DNA encoding an enzyme relating to the synthesis ofthe intracellular sugar nucleotide, GDP-fucose or an enzyme relating tothe modification of a sugar chain in which 1-position of fucose is boundto 6-position of N-acetylglucosamine in the reducing terminal throughα-bond in a complex type N-glycoside-linked sugar chain is prepared.

The nucleotide sequence of the prepared cDNA or genomic DNA isdetermined.

Based on the determined DNA sequence, an antisense gene or a ribozyme ofappropriate length is designed which comprises a DNA moiety encoding theenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainin which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain, non-translated regions or introns.

In order to express the antisense gene or ribozyme in a cell, arecombinant vector is prepared by inserting a fragment or full-length ofthe prepared DNA into a site downstream of a promoter in an appropriateexpression vector.

A transformant can be obtained by introducing the recombinant vectorinto a host cell suited for the expression vector.

The host cell used for the production of the recombinant antibodycomposition comprising an antibody molecule having complex typeN-glycoside-linked sugar chains in the Fc region, wherein the ratio ofsugar chains in which fucose is not bound to N-acetylglucosamine in thereducing terminal of the sugar chains among the total complex typeN-glycoside-linked sugar chains which bind to the Fc region contained inthe composition is 20% or more of the present invention can be obtainedby selecting a transformant using, as an index, the activity of theenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainin which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain. The host cell used for theproduction of the high ADCC activity antibody-producing cell can also beobtained by selecting a transformant using, as an index, the sugar chainstructure of a glycoprotein on the cell membrane or the sugar chainstructure of the produced antibody molecule.

As the host cell used for the production of the high ADCC activityantibody-producing cell, any yeast, animal cell, insect cell, plantcell, or the like can be used so long as it has a gene encoding theenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainin which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain. Examples of the host cells includethose described in the above 1.

The expression vectors that can be employed are those capable ofautonomous replication or integration into the chromosome in the abovehost cells and comprising a promoter at a position appropriate for thetranscription of the designed antisense gene or ribozyme. Examples ofthe expression vectors include those described in the above 1.

Introduction of a gene into various host cells can be carried out by themethods suitable for introducing a recombinant vector into various hostcells described in the above 1.

Selection of a transformant using, as an index, the activity of anenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or an enzyme relating to the modification of a sugar chain inwhich 1-position of fucose is bound to 6-position of N-acetylglucosaminein the reducing terminal through α-bond in a complex typeN-glycoside-linked sugar chain can be carried out, for example, by thefollowing methods.

Methods for Selecting a Transformant

A cell in which the activity of an enzyme relating to the synthesis ofthe intracellular sugar nucleotide, GDP-fucose or an enzyme relating tothe modification of a sugar chain in which 1-position of fucose is boundto 6-position of N-acetylglucosamine in the reducing terminal throughα-bond in a complex type N-glycoside-linked sugar chain is deleted canbe selected by measuring the activity of the enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose or the enzymerelating to the modification of a sugar chain in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducingterminal through α-bond in a complex type N-glycoside-linked sugar chainusing biochemical methods or genetic engineering techniques described inShin Seikagaku Jikken Koza (New Lectures on Experiments in Biochemistry)3—Saccharides I, Glycoprotein (Tokyo Kagaku Dojin), edited by TheJapanese Biochemical Society (1988); Cell Technology, Extra Edition,Experimental Protocol Series, Glycobiology Experimental Protocol,Glycoprotein, Glycolipid and Proteoglycan (Shujunsha), edited by NaoyukiTaniguchi, Akemi Suzuki, Kiyoshi Furukawa and Kazuyuki Sugawara (1996);Molecular Cloning, Second Edition; Current Protocols in MolecularBiology; and the like. An example of the biochemical methods is a methodin which the enzyme activity is evaluated using an enzyme-specificsubstrate. Examples of the genetic engineering techniques includeNorthern analysis and RT-PCR in which the amount of mRNA for a geneencoding the enzyme is measured.

Selection of a transformant using, as an index, the sugar chainstructure of a glycoprotein on the cell membrane can be carried out, forexample, by the method described in 2(5) below. Selection of atransformant using, as an index, the sugar chain structure of a producedantibody molecule can be carried out, for example, by the methodsdescribed in 4 or 5 below.

Preparation of a cDNA encoding an enzyme relating to the synthesis of anintracellular sugar nucleotide, GDP-fucose or an enzyme relating to themodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing terminal throughα-bond in a complex type N-glycoside-linked sugar chain can be carriedout, for example, by the following method.

Preparation Method of cDNA

Total RNA or mRNA is prepared from a various host cell tissue or cell.

A cDNA library is prepared from the obtained total RNA or mRNA.

Degenerative primers are prepared based on the amino acid sequence of anenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or an enzyme relating to the modification of a sugar chain inwhich 1-position of fucose is bound to 6-position of N-acetylglucosaminein the reducing terminal through α-bond in a complex typeN-glycoside-linked sugar chain, and a gene fragment encoding the enzymerelating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainin which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain is obtained by PCR using theprepared cDNA library as a template.

A DNA encoding the enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose or the enzyme relating to the modificationof a sugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain can be obtained by screening thecDNA library using the obtained gene fragment as a probe.

As the mRNA of a human or non-human animal tissue or cell, commerciallyavailable one (for example, manufactured by Clontech) may be used, or itmay be prepared from a human or non-human animal tissue or cell in thefollowing manner.

The methods for preparing total RNA from a human or non-human animaltissue or cell include the guanidine thiocyanate-cesium trifluoroacetatemethod [Methods in Enzymology, 154, 3 (1987)], the acidic guanidinethiocyanate-phenol-chloroform (AGPC) method [Analytical Biochemistry,162, 156 (1987); Experimental Medicine, 9, 1937 (1991)] and the like.

The methods for preparing mRNA as poly(A)⁺RNA from the total RNA includethe oligo (dT) immobilized cellulose column method (Molecular Cloning,Second Edition).

It is also possible to prepare mRNA by using a commercially availablekit such as Fast Track mRNA Isolation Kit (manufactured by Invitrogen)or Quick Prep™ mRNA Purification Kit (manufactured by Pharmacia).

A cDNA library is prepared from the obtained mRNA of a human ornon-human animal tissue or cell. The methods for preparing the cDNAlibrary include the methods described in Molecular Cloning, SecondEdition; Current Protocols in Molecular Biology; A Laboratory Manual,2nd Ed.(1989); etc., and methods using commercially available kits suchas SuperScript™ Plasmid System for cDNA Synthesis and Plasmid Cloning(manufactured by Life Technologies) and ZAP-cDNA™ Synthesis Kit(manufactured by STRATAGENE).

As the cloning vector for preparing the cDNA library, any vectors, e.g.phage vectors and plasmid vectors, can be used so long as they areautonomously replicable in Escherichia coli K12. Examples of suitablevectors include ZAP™ Express [manufactured by STRATAGENE; Strategies, 5,58 (1992)], pBluescript II SK(+) [Nucleic Acids Research, 17, 9494(1989)], λZAP II (manufactured by STRATAGENE), λgt10, λgt11 [DNACloning, A Practical Approach, 1, 49 (1985)], λTriplEx (manufactured byClontech), λExCell (manufactured by Pharmacia), pT7T318U (manufacturedby Pharmacia), pcD2 [Mol. Cell. Biol., 3, 280 (1983)], pUC18 [Gene, 33,103 (1985)], and the like.

Any microorganism can be used as the host microorganism for preparingthe cDNA library, but Escherichia coli is preferably used. Examples ofsuitable host microorganisms are Escherichia coli XL1-Blue MRF′[manufactured by STRATAGENE; Strategies, 5, 81 (1992)], Escherichia coliC600 [Genetics, 39, 440 (1954)], Escherichia coli Y1088 [Science, 222,778 (1983)], Escherichia coli Y1090 [Science, 222, 778 (1983)],Escherichia coli NM522 [J. Mol. Biol., 166, 1 (1983)], Escherichia coliK802 [J. Mol. Biol., 16, 118 (1966)], Escherichia coli JM105 [Gene, 38,275 (1985)], and the like.

The cDNA library may be used as such in the following analysis.Alternatively, in order to efficiently obtain full-length cDNAs bydecreasing the ratio of partial cDNAs, a cDNA library prepared using theoligo-cap method developed by Sugano, et al. [Gene, 138, 171 (1994);Gene, 200, 149 (1997); Protein, Nucleic Acid and Enzyme, 41, 603 (1996);Experimental Medicine, 11, 2491 (1993); cDNA Cloning (Yodosha) (1996);Methods for Preparing Gene Libraries (Yodosha) (1994)] may be used inthe following analysis.

Degenerative primers specific for the 5′-terminal and 3′-terminalnucleotide sequences of a nucleotide sequence presumed to encode theamino acid sequence of an enzyme relating to the synthesis of anintracellular sugar nucleotide, GDP-fucose or an enzyme relating to themodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing terminal throughα-bond in a complex type N-glycoside-linked sugar chain are preparedbased on the amino acid sequence of the enzyme. A gene fragment encodingthe enzyme relating to the synthesis of an intracellular sugarnucleotide, GDP-fucose or the enzyme relating to the modification of asugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain can be obtained by DNA amplificationby PCR [PCR Protocols, Academic Press (1990)] using the prepared cDNAlibrary as a template.

It can be confirmed that the obtained gene fragment is a DNA encodingthe enzyme relating to the synthesis of an intracellular sugarnucleotide, GDP-fucose or the enzyme relating to the modification of asugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain by analyzing the nucleotide sequenceby generally employed nucleotide sequence analyzing methods such as thedideoxy method of Sanger, et al. [Proc. Natl. Acad. Sci. U.S.A., 74,5463 (1977)] or by use of nucleotide sequence analyzers such as ABIPRISM 377 DNA Sequencer (manufactured by Applied Biosystems).

A DNA encoding the enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose or the enzyme relating to the modificationof a sugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain can be obtained from the cDNA orcDNA library synthesized from the mRNA contained in a human or non-humananimal tissue or cell by colony hybridization or plaque hybridization(Molecular Cloning, Second Edition) using the above gene fragment as aprobe.

A cDNA encoding the enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose or the enzyme relating to the modificationof a sugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain can also be obtained byamplification by PCR using the cDNA or cDNA library synthesized from themRNA contained in a human or non-human animal tissue or cell as atemplate and using the primers used for obtaining the gene fragmentencoding the enzyme relating to the synthesis of an intracellular sugarnucleotide, GDP-fucose or the enzyme relating to the modification of asugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain.

The nucleotide sequence of the DNA encoding the enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose or the enzymerelating to the modification of a sugar chain in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducingterminal through α-bond in a complex type N-glycoside-linked sugar chaincan be determined by generally employed nucleotide sequence analyzingmethods such as the dideoxy method of Sanger, et al. [Proc. Natl. Acad.Sci. U.S.A., 74, 5463 (1977)] or by use of nucleotide sequence analyzerssuch as ABI PRISM 377 DNA Sequencer™ (manufactured by AppliedBiosystems).

By carrying out a search of nucleotide sequence databases such asGenBank, EMBL or DDBJ using a homology search program such as BLASTbased on the determined nucleotide sequence of the cDNA, it can beconfirmed that the obtained DNA is a gene encoding the enzyme relatingto the synthesis of an intracellular sugar nucleotide, GDP-fucose or theenzyme relating to the modification of a sugar chain in which 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingterminal through α-bond in a complex type N-glycoside-linked sugar chainamong the genes in the nucleotide sequence database.

Examples of the nucleotide sequences of the genes encoding the enzymerelating to the synthesis of an intracellular sugar nucleotide,GDP-fucose obtained by the above methods include the nucleotidesequences represented by SEQ ID NO:18 or 20.

Examples of the nucleotide sequences of the genes encoding the enzymerelating to the modification of a sugar chain in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducingterminal through α-bond in a complex type N-glycoside-linked sugar chainobtained by the above methods include the nucleotide sequencerepresented by SEQ ID NO:22 or 23.

The cDNA encoding the enzyme relating to the synthesis of anintracellular sugar nucleotide, GDP-fucose or the enzyme relating to themodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing terminal throughα-bond in a complex type N-glycoside-linked sugar chain can also beobtained by chemical synthesis with a DNA synthesizer such as DNASynthesizer Model 392 (manufactured by Perkin Elmer) utilizing thephosphoamidite method based on the determined nucleotide sequence of thedesired DNA.

Preparation of a genomic DNA encoding the enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose or the enzymerelating to the modification of a sugar chain in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducingterminal through α-bond in a complex type N-glycoside-linked sugar chaincan be carried out, for example, by the following method.

Method for Preparing Genomic DNA

The genomic DNA can be prepared by known methods described in MolecularCloning, Second Edition, Current Protocols in Molecular Biology, etc. Inaddition, the genomic DNA encoding the enzyme relating to the synthesisof an intracellular sugar nucleotide, GDP-fucose or the enzyme relatingto the modification of a sugar chain in which 1-position of fucose isbound to 6-position of N-acetylglucosamine in the reducing terminalthrough α-bond in a complex type N-glycoside-linked sugar chain can alsobe obtained by using a kit such as Genomic DNA Library Screening System(manufactured by Genome Systems) or Universal GenomeWalker™ Kits(manufactured by CLONTECH).

The nucleotide sequence of the DNA encoding the enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose or the enzymerelating to the modification of a sugar chain in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducingterminal through α-bond in a complex type N-glycoside-linked sugar chaincan be determined by generally employed nucleotide analyzing methodssuch as the dideoxy method of Sanger, et al. [Proc. Natl. Acad. Sci.U.S.A., 74, 5463 (1977)] or by use of nucleotide sequence analyzers suchas ABI PRISM 377 DNA Sequencer (manufactured by Applied Biosystems).

By carrying out a search of nucleotide sequence databases such asGenBank, EMBL or DDBJ using a homology search program such as BLASTbased on the determined nucleotide sequence of the genomic DNA, it canbe confirmed that the obtained DNA is a gene encoding the enzymerelating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainin which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain among the genes in the nucleotidesequence database.

The genomic DNA encoding the enzyme relating to the synthesis of anintracellular sugar nucleotide, GDP-fucose or the enzyme relating to themodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing terminal throughα-bond in a complex type N-glycoside-linked sugar chain can also beobtained by chemical synthesis with a DNA synthesizer such as DNASynthesizer Model 392 (manufactured by Perkin Elmer) utilizing thephosphoamidite method based on the determined nucleotide sequence of theDNA.

Examples of the nucleotide sequences of the genomic DNAs encoding theenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose obtained by the above methods include the nucleotidesequences represented by SEQ ID NOs:26, 27, 28 and 29.

An example of the nucleotide sequence of the genomic DNA encoding theenzyme relating to the modification of a sugar chain in which 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingterminal through α-bond in a complex type N-glycoside-linked sugar chainobtained by the above methods is the nucleotide sequence represented bySEQ ID NO:30.

The host cell used for the production of the antibody composition of thepresent invention can also be obtained without using an expressionvector by directly introducing into a host cell an antisenseoligonucleotide or ribozyme designed based on the nucleotide sequenceencoding the enzyme relating to the synthesis of an intracellular sugarnucleotide, GDP-fucose or the enzyme relating to the modification of asugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain.

The antisense oligonucleotide or ribozyme can be prepared by knownmethods or by using a DNA synthesizer. Specifically, based on thesequence information on an oligonucleotide having a sequencecorresponding to 5 to 150, preferably 5 to 60, more preferably 10 to 40continuous nucleotides in the nucleotide sequence of the cDNA andgenomic DNA encoding the enzyme relating to the synthesis of anintracellular sugar nucleotide, GDP-fucose or the enzyme relating to themodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing terminal throughα-bond in a complex type N-glycoside-linked sugar chain, anoligonucleotide corresponding to the sequence complementary to the aboveoligonucleotide (antisense oligonucleotide) or a ribozyme comprising theoligonucleotide sequence can be synthesized.

The oligonucleotide includes oligo RNA and derivatives of theoligonucleotide (hereinafter referred to as oligonucleotidederivatives).

The oligonucleotide derivatives include an oligonucleotide derivativewherein the phosphodiester bond in the oligonucleotide is converted to aphosophorothioate bond, an oligonucleotide derivative wherein thephosphodiester bond in the oligonucleotide is converted to an N3′-P5′phosphoamidate bond, an oligonucleotide derivative wherein theribose-phosphodiester bond in the oligonucleotide is converted to apeptide-nucleic acid bond, an oligonucleotide derivative wherein theuracil in the oligonucleotide is substituted with C-5 propynyluracil, anoligonucleotide derivative wherein the uracil in the oligonucleotide issubstituted with C-5 thiazolyluracil, an oligonucleotide derivativewherein the cytosine in the oligonucleotide is substituted with C-5propynylcytosine, an oligonucleotide derivative wherein the cytosine inthe oligonucleotide is substituted with phenoxazine-modified cytosine,an oligonucleotide derivative wherein the ribose in the oligonucleotideis substituted with 2′-O-propylribose, and an oligonucleotide derivativewherein the ribose in the oligonucleotide is substituted with2′-methoxyethoxyribose [Cell Technology, 16, 1463 (1997)].

(b) Preparation of the Host Cell for the Production of High ADCCActivity Antibody-Producing Cell by the Homologous Recombination Method

The host cell used for the production of the high ADCC activityantibody-producing cell of the present invention can be prepared bymodifying a target gene on the chromosome by the homologousrecombination method targeting a gene encoding an enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose or an enzymerelating to the modification of a sugar chain in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducingterminal through α-bond in a complex type N-glycoside-linked sugarchain.

Modification of the target gene on the chromosome can be carried out byusing the methods described in Manipulating the Mouse Embryo, ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press(1994) (hereinafter referred to as “Manipulating the Mouse Embryo, ALaboratory Manual”; Gene Targeting, A Practical Approach, IRL Press atOxford University Press (1993); Biomanual Series 8, Gene Targeting,Preparation of Mutant Mice Using ES Cells, Yodosha (1995) (hereinafterreferred to as Preparation of Mutant Mice Using ES Cells); etc., forexample, in the following manner.

A genomic DNA encoding an enzyme relating to the synthesis of anintracellular sugar nucleotide, GDP-fucose or an enzyme relating to themodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing terminal throughα-bond in a complex type N-glycoside-linked sugar chain is prepared.

Based on the nucleotide sequence of the genomic DNA, a target vector isprepared for homologous recombination of a target gene to be modified(e.g., the structural gene or promoter gene for the enzyme relating tothe synthesis of an intracellular sugar nucleotide, GDP-fucose or theenzyme relating to the modification of a sugar chain in which 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingterminal through α-bond in a complex type N-glycoside-linked sugarchain).

The host cell used for the production of the high ADCC activityantibody-producing cell can be prepared by introducing the preparedtarget vector into a host cell and selecting a cell in which homologousrecombination occurred between the target gene on the chromosome and thetarget vector.

As the host cell, any yeast, animal cell, insect cell, plant cell, orthe like can be used so long as it has a gene encoding the enzymerelating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainin which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain. Examples of the host cells includethose described in the above 1.

The genomic DNA encoding the enzyme relating to the synthesis of anintracellular sugar nucleotide, GDP-fucose or the enzyme relating to themodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing terminal throughα-bond in a complex type N-glycoside-linked sugar chain can be preparedby the methods for preparing a genomic DNA described in the above 1 (1)(a).

Examples of the nucleotide sequences of the genomic DNAs encoding theenzyme relating to the synthesis of the intracellular sugar nucleotide,GDP-fucose obtained by the above methods include the nucleotidesequences represented by SEQ ID NOs:26, 27, 28 and 29.

An example of the nucleotide sequence of the genomic DNA encoding theenzyme relating to the modification of a sugar chain in which 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingterminal through α-bond in a complex type N-glycoside-linked sugar chainobtained by the above methods is the nucleotide sequence represented bySEQ ID NO:30.

The target vector for use in the homologous recombination of the targetgene on the chromosome can be prepared according to the methodsdescribed in Gene Targeting, A Practical Approach, IRL Press at OxfordUniversity Press (1993); Biomanual Series 8, Gene Targeting, Preparationof Mutant Mice Using ES Cells, Yodosha (1995); etc. The target vectormay be either a replacement-type or an insertion-type.

Introduction of the target vector into various host cells can be carriedout by the methods suitable for introducing a recombinant vector intovarious host cells described in the above 1.

The methods for efficiently selecting a homologous recombinant includepositive selection, promoter selection, negative selection and polyAselection described in Gene Targeting, A Practical Approach, IRL Pressat Oxford University Press (1993); Biomanual Series 8, Gene Targeting,Preparation of Mutant Mice Using ES Cells, Yodosha (1995); etc. Themethods for selecting the desired homologous recombinant from theselected cell lines include Southern hybridization (Molecular Cloning,Second Edition) and PCR [PCR Protocols, Academic Press (1990)] with thegenomic DNA.

(c) Preparation of the Host Cell for the High ADCC ActivityAntibody-Producing Cell by the RDO Method

The host cell used for the production of the high ADCC activityantibody-producing cell can be prepared by the RDO method targeting agene encoding an enzyme relating to the synthesis of the intracellularsugar nucleotide, GDP-fucose or an enzyme relating to the modificationof a sugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain, for example, in the followingmanner.

A cDNA or a genomic DNA encoding an enzyme relating to the synthesis ofthe intracellular sugar nucleotide, GDP-fucose or an enzyme relating tothe modification of a sugar chain in which 1-position of fucose is boundto 6-position of N-acetylglucosamine in the reducing terminal throughα-bond in a complex type N-glycoside-linked sugar chain is prepared bythe methods described in the above 1 (1) (a).

The nucleotide sequence of the prepared cDNA or genomic DNA isdetermined.

Based on the determined DNA sequence, an RDO construct of appropriatelength which comprises a part encoding the enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose or the enzymerelating to the modification of a sugar chain in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducingterminal through α-bond in a complex type N-glycoside-linked sugarchain, a part of its non-translated region or a part of introns isdesigned and synthesized.

The host cell of the present invention can be obtained by introducingthe synthesized RDO into a host cell and then selecting a transformantin which a mutation occurred in the target enzyme, that is, the enzymerelating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainin which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain.

As the host cell, any yeast, animal cell, insect cell, plant cell, orthe like can be used so long as it has a gene encoding the enzymerelating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainin which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain. Examples of the host cells includethose described in the above 1.

Introduction of the RDO into various host cells can be carried out bythe methods suitable for introducing a recombinant vector into varioushost cells described in the above 1.

The cDNA encoding the enzyme relating to the synthesis of anintracellular sugar nucleotide, GDP-fucose or the enzyme relating to themodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing terminal throughα-bond in a complex type N-glycoside-linked sugar chain can be preparedby the methods for preparing a cDNA described in the above 2 (1) (a) orthe like.

The genomic DNA encoding the enzyme relating to the synthesis of anintracellular sugar nucleotide, GDP-fucose or the enzyme relating to themodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing terminal throughα-bond in a complex type N-glycoside-linked sugar chain can be preparedby the methods for preparing a genomic DNA described in the above 2 (1)(b) or the like.

After DNA is cleaved with appropriate restriction enzymes, thenucleotide sequence of the DNA can be determined by subcloning the DNAfragments into a plasmid such as pBluescript™ SK(−) (manufactured byStratagene), subjecting the clones to the reaction generally used as amethod for analyzing a nucleotide sequence such as the dideoxy method ofSanger et al. [Proc. Natl. Acad. Sci., USA, 74, 5463 (1977)] or thelike, and then analyzing the clones by using an automatic nucleotidesequence analyzer such as ABI PRISM 377 DNA Sequencer™ (manufactured byApplied Biosystems) or the like.

The RDO can be prepared by conventional methods or by using a DNAsynthesizer.

The methods for selecting a cell in which a mutation occurred byintroducing the RDO into the host cell, in the gene encoding the enzyme,that is, the enzyme relating to the synthesis of an intracellular sugarnucleotide, GDP-fucose or the enzyme relating to the modification of asugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain include the methods for directlydetecting mutations in chromosomal genes described in Molecular Cloning,Second Edition, Current Protocols in Molecular Biology, and the like.

For the selection of the transformant, the following methods can also beemployed: the method using, as an index, the activity of the enzymerelating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainin which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain described in the above 2 (1) (a);the method using, as an index, the sugar chain structure of aglycoprotein on the cell membrane described in 2 (5) below; and themethod using, as an index, the sugar chain structure of a producedantibody molecule described in 4 or 5 below.

The RDO can be designed according to the descriptions in Science, 273,1386 (1996); Nature Medicine, 4, 285 (1998); Hepatology, 25, 1462(1997); Gene Therapy, 5, 1960 (1999); Gene Therapy, 5, 1960 (1999); J.Mol. Med., 75, 829 (1997); Proc. Natl. Acad. Sci. USA, 96, 8774 (1999);Proc. Natl. Acad. Sci. USA, 96, 8768 (1999); Nuc. Acids Res., 27, 1323(1999); Invest. Dermatol., 111, 1172 (1998); Nature Biotech., 16, 1343(1998); Nature Biotech., 18, 43 (2000); Nature Biotech., 18, 555 (2000);and the like.

(d) Preparation of the Host Cell for the Production of the High ADCCActivity Antibody-Producing Cell by the RNAi Method

The host cell used for the production of the high ADCC activityantibody-producing cell can be prepared by the RNAi method targeting agene encoding an enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose or an enzyme relating to the modificationof a sugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain, for example, in the followingmanner.

A cDNA encoding an enzyme relating to the synthesis of the intracellularsugar nucleotide, GDP-fucose or an enzyme relating to the modificationof a sugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain is prepared by the methods describedin the above 2 (1) (a).

The nucleotide sequence of the prepared cDNA is determined.

Based on the determined cDNA sequence, an RNAi gene of appropriatelength is designed which comprises a part encoding the enzyme relatingto the synthesis of an intracellular sugar nucleotide, GDP-fucose or theenzyme relating to the modification of a sugar chain in which 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingterminal through α-bond in a complex type N-glycoside-linked sugarchain, or a part of non-translated regions.

In order to express the RNAi gene in a cell, a recombinant vector isprepared by inserting a fragment or full-length of the prepared cDNAinto a site downstream of a promoter in an appropriate expressionvector.

The recombinant vector is introduced into a host cell suited for theexpression vector to obtain a transformant.

The host cell used for the preparation of the host cell can be obtainedby selecting a transformant using, as an index, the activity of theenzyme relating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainin which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain, or the sugar chain structure of aproduced antibody molecule or a glycoprotein on the cell membrane.

As the host cell, any yeast, animal cell, insect cell, plant cell, orthe like can be used so long as it has a gene encoding the enzymerelating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainin which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain. Examples of the host cells includethose described in the above 1.

The expression vectors that can be employed are those capable ofautonomous replication or integration into the chromosome in the abovehost cells and comprising a promoter at a position appropriate for thetranscription of the designed RNAi gene. Examples of the expressionvectors include those described in the above 1.

Introduction of a gene into various host cells can be carried out by themethods suitable for introducing a recombinant vector into various hostcells described in the above 1.

The methods for selecting the transformant using, as an index, theactivity of the enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose or the activity of the enzyme relating tothe modification of a sugar chain in which 1-position of fucose is boundto 6-position of N-acetylglucosamine in the reducing terminal throughα-bond in a complex type N-glycoside-linked sugar chain include themethods described in the above 2 (1) (a).

The methods for selecting the transformant using, as an index, the sugarchain structure of a glycoprotein on the cell membrane include themethod described in 2 (5). The methods for selecting the transformantusing, as an index, the sugar chain structure of a produced antibodymolecule include the methods described in 4 or 5 below.

The methods for preparing cDNA encoding the enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose or the enzymerelating to the modification of a sugar chain in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducingterminal through α-bond in a complex type N-glycoside-linked sugar chaininclude the methods for preparing a cDNA described in the above 2 (1)(a), and the like.

The host cell used for the production of the high CDC activity and highADCC activity antibody-producing cell of the present invention can alsobe obtained, without using an expression vector, by directly introducinginto a host cell the RNAi gene designed based on the nucleotide sequenceencoding the enzyme relating to the synthesis of an intracellular sugarnucleotide, GDP-fucose or the enzyme relating to the modification of asugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain.

The RNAi gene can be prepared by known methods or by using a DNAsynthesizer.

The RNAi gene construct can be designed according to the descriptions inNature, 391, 806 (1998); Proc. Natl. Acad. Sci. USA, 95, 15502 (1998);Nature, 395, 854 (1998); Proc. Natl. Acad. Sci. USA, 96, 5049 (1999);Cell, 95, 1017 (1998); Proc. Natl. Acad. Sci. USA, 96, 1451 (1999);Proc. Natl. Acad. Sci. USA, 95, 13959 (1998); Nature Cell Biol., 2, 70(2000); and the like.

(e) Preparation of the Host Cell for the Production of the High ADCCActivity Antibody-Producing Cell by the Method Using a Transposon

The host cell used for the production of the high ADCC activityantibody-producing cell can be prepared by using the transposon systemdescribed in Nature Genet., 25, 35 (2000), and the like, and thenselecting a mutant using, as an index, the activity of the enzymerelating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the activity of the enzyme relating to the modification ofa sugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain, or the sugar chain structure of aproduced antibody molecule or a glycoprotein on the cell membrane.

The transposon system is a system for inducing a mutation by randominsertion of an exogenous gene into the chromosome, wherein usually anexogenous gene inserted into a transposon is used as a vector forinducing a mutation and a transposase expression vector for randomlyinserting the gene into the chromosome is introduced into the cell atthe same time.

Any transposase can be used so long as it is suitable for the sequenceof the transposon to be used.

As the exogenous gene, any gene can be used so long as it can induce amutation in the DNA of a host cell.

As the host cell, any yeast, animal cell, insect cell, plant cell, orthe like can be used so long as it has a gene encoding the enzymerelating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainin which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain. Examples of the host cells includethose described in the above 1. Introduction of the gene into varioushost cells can be carried out by the methods suitable for introducing arecombinant vector into various host cells described in the above 1.

The methods for selecting the mutant using, as an index, the activity ofthe enzyme relating to the synthesis of an intracellular sugarnucleotide, GDP-fucose or the enzyme relating to the modification of asugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain include the methods described in theabove 2 (1) (a).

The methods for selecting the mutant using, as an index, the sugar chainstructure of a glycoprotein on the cell membrane include the methoddescribed in 2 (5). The methods for selecting the mutant using, as anindex, the sugar chain structure of a produced antibody molecule includethe methods described in 4 or 5 below.

(2) Technique of Introducing a Dominant-Negative Mutant of a GeneEncoding an Enzyme

The host cell used for the production of the high ADCC activityantibody-producing cell can be prepared by using the technique ofintroducing a dominant-negative mutant of a target gene, i.e., a geneencoding an enzyme relating to the synthesis of the intracellular sugarnucleotide, GDP-fucose or an enzyme relating to the modification of asugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain. Examples of the enzymes relating tothe synthesis of the intracellular sugar nucleotide, GDP-fucose includeGMD and Fx. Examples of the enzymes relating to the modification of asugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain include a1,6-fucosyltransferase andα-L-fucosidase.

These enzymes have substrate specificity and catalyze specificreactions. By disrupting the active center of such enzymes havingsubstrate specificity and catalytic activity, their dominant-negativemutants can be prepared. Preparation of a dominant-negative mutant isdescribed in detail below, using GMD as an example among the targetenzymes.

As a result of the analysis of the three-dimensional structure of GMDderived from Escherichia coli, it has been revealed that four aminoacids (threonine at position 133, glutamic acid at position 135,tyrosine at position 157 and lysine at position 161) have an importantfunction for the enzyme activity (Structure, 8, 2, 2000). That is, themutants prepared by substituting the above four amino acids by otheramino acids based on the three-dimensional structure information allshowed significantly decreased enzyme activity. On the other hand,little change was observed in the ability of the mutants to bind to theGMD coenzyme NADP or the substrate GDP-mannose. Accordingly, adominant-negative mutant can be prepared by substituting the four aminoacids which are responsible for the enzyme activity of GMD. On the basisof the result of preparation of a dominant-negative mutant of GMDderived from Escherichia coli, dominant-negative mutants can be preparedby performing homology comparison and three-dimensional structureprediction using the amino acid sequence information. For example, inthe case of GMD derived from CHO cell (SEQ ID NO:19), adominant-negative mutant can be prepared by substituting threonine atposition 155, glutamic acid at position 157, tyrosine at position 179and lysine at position 183 bp other amino acids. Preparation of such agene carrying introduced amino acid substitutions can be carried out bysite-directed mutagenesis described in Molecular Cloning, SecondEdition, Current Protocols in Molecular Biology, and the like.

The host cell used for the production of the high ADCC activityantibody-producing cell can be prepared according to the method of geneintroduction described in Molecular Cloning, Second Edition, CurrentProtocols in Molecular Biology, Manipulating the Mouse Embryo, SecondEdition, and the like using a gene encoding a dominant-negative mutantof a target enzyme (hereinafter abbreviated as dominant-negative mutantgene) prepared as above, for example, in the following manner.

A dominant-negative mutant gene encoding the enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose or the enzymerelating to the modification of a sugar chain in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducingterminal through α-bond in a complex type N-glycoside-linked sugar chainis prepared.

Based on the full-length DNA of the prepared dominant-negative mutantgene, a DNA fragment of appropriate length containing a region encodingthe protein is prepared according to need.

A recombinant vector is prepared by inserting the DNA fragment orfull-length DNA into a site downstream of a promoter in an appropriateexpression vector.

The recombinant vector is introduced into a host cell suited for theexpression vector to obtain a transformant.

The host cell used for the preparation of the high ADCC activityantibody-producing cell can be obtained by selecting a transformantusing, as an index, the activity of the enzyme relating to the synthesisof an intracellular sugar nucleotide, GDP-fucose or the enzyme relatingto the modification of a sugar chain in which 1-position of fucose isbound to 6-position of N-acetylglucosamine in the reducing terminalthrough α-bond in a complex type N-glycoside-linked sugar chain, or thesugar chain structure of a produced antibody molecule or a glycoproteinon the cell membrane.

As the host cell, any yeast, animal cell, insect cell, plant cell, orthe like can be used so long as it has a gene encoding the enzymerelating to the synthesis of an intracellular sugar nucleotide,GDP-fucose or the enzyme relating to the modification of a sugar chainin which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in a complextype N-glycoside-linked sugar chain. Examples of the host cells includethose described in the above 1.

The expression vectors that can be employed are those capable ofautonomous replication or integration into the chromosome in the abovehost cells and comprising a promoter at a position appropriate for thetranscription of the DNA encoding the desired dominant-negative mutant.Examples of the expression vectors include those described in the above1.

Introduction of a gene into various host cells can be carried out by themethods suitable for introducing a recombinant vector into various hostcells described in the above 1.

The methods for selecting the transformant using, as an index, theactivity of the enzyme relating to the synthesis of an intracellularsugar nucleotide, GDP-fucose or the activity of the enzyme relating tothe modification of a sugar chain in which 1-position of fucose is boundto 6-position of N-acetylglucosamine in the reducing terminal throughα-bond in a complex type N-glycoside-linked sugar chain include themethods described in 2 (1) (a) below.

The methods for selecting the transformant using, as an index, the sugarchain structure of a glycoprotein on the cell membrane include themethod described in 2 (5) below. The methods for selecting thetransformant using, as an index, the sugar chain structure of a producedantibody molecule include the methods described in 4 or 5 below.

(3) Technique of Introducing a Mutation into an Enzyme

The host cell used for the high ADCC activity antibody-producing cellcan be prepared by introducing a mutation into a gene encoding an enzymerelating to the synthesis of the intracellular sugar nucleotide,GDP-fucose or an enzyme relating to the modification of a sugar chain inwhich 1-position of fucose is bound to 6-position of N-acetylglucosaminein the reducing terminal through α-bond in a complex typeN-glycoside-linked sugar chain, and then selecting a desired cell linein which the mutation occurred in the enzyme.

Examples of the enzymes relating to the synthesis of the intracellularsugar nucleotide, GDP-fucose include GMD, Fx, and the like. Examples ofthe enzymes relating to the modification of a sugar chain in which1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing terminal through α-bond in a complex typeN-glycoside-linked sugar chain include α1,6-fucosyltransferase,α-L-fucosidase, and the like.

The methods for introducing a mutation into the enzyme include: 1) amethod in which a desired cell line is selected from mutants obtained bysubjecting a parent cell line to mutagenesis or by spontaneous mutationusing, as an index, the activity of the enzyme relating to the synthesisof an intracellular sugar nucleotide, GDP-fucose or the activity of theenzyme relating to the modification of a sugar chain in which 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingterminal through α-bond in a complex type N-glycoside-linked sugarchain; 2) a method in which a desired cell line is selected from mutantsobtained by subjecting a parent cell line to mutagenesis or byspontaneous mutation using, as an index, the sugar chain structure of aproduced antibody molecule; and 3) a method in which a desired cell lineis selected from mutants obtained by subjecting a parent cell line tomutagenesis or by spontaneous mutation using, as an index, the sugarchain structure of a glycoprotein on the cell membrane.

Mutagenesis may be carried out by any method capable of inducing a pointmutation, a deletion mutation or a frameshift mutation in DNA of a cellof a parent cell line.

Suitable methods include treatment with ethyl nitrosourea,nitrosoguanidine, benzopyrene or an acridine dye and irradiation.Various alkylating agents and carcinogens are also useful as mutagens. Amutagen is allowed to act on a cell by the methods described in SoshikiBaiyo no Gijutsu (Tissue Culture Techniques), Third Edition (AsakuraShoten), edited by The Japanese Tissue Culture Association (1996);Nature Genet., 24, 314 (2000); and the like.

Examples of the mutants generated by spontaneous mutation includespontaneous mutants obtained by continuing subculture under usual cellculture conditions without any particular treatment for mutagenesis.

The methods for measuring the activity of the enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose or the enzymerelating to the modification of a sugar chain in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducingterminal through α-bond in a complex type N-glycoside-linked sugar chaininclude the methods described in the above 1 (1) (a). The methods fordetermining the sugar chain structure of a produced antibody moleculeinclude the methods described in 4 or 5 below. The methods fordetermining the sugar chain structure of a glycoprotein on the cellmembrane include the method described in the above 2 (5).

(4) Technique of Suppressing Transcription or Translation of a GeneEncoding an Enzyme

The host cell used for the production of the high ADCC activityantibody-producing cell can be prepared by suppressing transcription ortranslation of a target gene, i.e., a target gene encoding an enzymerelating to the synthesis of the intracellular sugar nucleotide,GDP-fucose or an enzyme relating to the modification of a sugar chain inwhich 1-position of fucose is bound to 6-position of N-acetylglucosaminein the reducing terminal through α-bond in a complex typeN-glycoside-linked sugar chain using the antisense RNA/DNA technique[Bioscience and Industry, 50, 322 (1992); Chemistry, 46, 681 (1991);Biotechnology, 9, 358 (1992); Trends in Biotechnology, 10, 87 (1992);Trends in Biotechnology, 10, 152 (1992); Cell Technology, 16, 1463(1997)], the triple helix technique [Trends in Biotechnology, 10, 132(1992)], and the like.

Examples of the enzymes relating to the synthesis of the intracellularsugar nucleotide, GDP-fucose include GMD, Fx, and the like. Examples ofthe enzymes relating to the modification of a sugar chain in which1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing terminal through α-bond in a complex typeN-glycoside-linked sugar chain include α1,6-fucosyltransferase,α-L-fucosidase, and the like.

The methods for measuring the activity of the enzyme relating to thesynthesis of an intracellular sugar nucleotide, GDP-fucose or theactivity of the enzyme relating to the modification of a sugar chain inwhich 1-position of fucose is bound to 6-position of N-acetylglucosaminein the reducing terminal through α-bond in a complex typeN-glycoside-linked sugar chain include the methods described in theabove 2 (1) (a).

The methods for determining the sugar chain structure of a glycoproteinon the cell membrane include the method described in the above 2 (5).The methods for determining the sugar chain structure of a producedantibody molecule include the methods described in 4 or 5 below.

(5) Technique of Selecting a Cell Line Resistant to a Lectin WhichRecognizes a Sugar Chain Structure in Which 1-Position of Fucose isBound to 6-Position of N-Acetylglucosamine in the Reducing TerminalThrough α-Bond in a N-Glycoside-Linked Sugar Chain

The host cell used for the production of the high ADCC activityantibody-producing cell can be prepared by selecting a cell lineresistant to a lectin which recognizes a sugar chain structure in which1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing terminal through α-bond in a N-glycoside-linked sugarchain.

Selection of a cell line resistant to a lectin which recognizes a sugarchain structure in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in aN-glycoside-linked sugar chain can be carried out, for example, by themethod using a lectin described in Somatic Cell Mol. Genet., 12, 51(1986), and the like.

As the lectin, any lectin can be used so long as it recognizes a sugarchain structure in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing terminal through α-bond in aN-glycoside-linked sugar chain. Specific examples include lentil lectinLCA (lentil agglutinin derived from Lens culinaris), pea lectin PSA (pealectin derived from Pisum sativum), broad bean lectin VFA (agglutininderived from Vicia faba) and Aleuria aurantia lectin AAL (lectin derivedfrom Aleuria aurantia).

Specifically, the cell line resistant to a lectin which recognizes asugar chain structure in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing terminal throughα-bond in a N-glycoside-linked sugar chain can be selected by culturingcells in a medium containing the above lectin at a concentration of 1μg/ml to 1 mg/ml for one day to 2 weeks, preferably one day to one week,subculturing surviving cells or picking up a colony and transferring itinto a culture vessel, and subsequently continuing the culturing usingthe medium containing the lectin.

3. Evaluation of the Activity of the Antibody Composition

The protein amount, antigen-binding activity or cytotoxic activity ofthe purified antibody composition can be measured using the knownmethods described in Monoclonal Antibodies, Antibody Engineering, or thelike.

Specifically, when the antibody composition is a human chimeric antibodyor a humanized antibody, the binding activity to an antigen or thebinding activity to cultured cell line which is antigen-positive can bemeasured by ELISA, the fluorescent antibody technique [Cancer Immunol.Immunother., 36, 373 (1993)], and the like. The cytotoxic activity tocultured cell line which is antigen-positive can be evaluated bymeasuring CDC activity, ADCC activity, or the like [Cancer Immunol.Immunother., 36, 373 (1993)].

The method for measuring ADCC activity includes a method in which atarget cell labeled with a radioisotope, a fluorescent substance, a dyeor the like is allowed to contact with an antibody and an effector cell,and then the activity of the labeled substance released from the injuredtarget cell is measured; a method in which a target cell is allowed tocontact with an antibody and an effector cell, and then the biologicalactivity of an enzyme released from the injured target cell is measured;and the like.

The method for measuring CDC activity includes a method in which atarget cell labeled with a radioisotope, a fluorescent substance, a dyeor the like is allowed to contact with an antibody and a biologicalspecimen such as serum containing a complement component, and then theactivity of the labeled substance released from the injured target cellis measured; a method in which a target cell is allowed to contact withan antibody and a biological specimen such as serum containing acomplement component, and then the biological activity of an enzymereleased from the injured target cell is measured; and the like.

The safety and therapeutic effect of the antibody composition in humancan be evaluated using an appropriate animal model of a speciesrelatively close to human, e.g., cynomolgus monkey.

4. Analysis of Sugar Chains in the Antibody Composition

The sugar chain structure of the antibody molecule expressed in variouscells can be analyzed according to general methods of analyzing thesugar chain structure of glycoprotein. For example, a sugar chain boundto an IgG molecule consists of neutral sugars such as galactose, mannoseand fucose, amino sugars such as N-acetylglucosamine, and acidic sugarssuch as sialic acid, and can be analyzed by techniques such as sugarcomposition analysis and sugar chain structure analysis usingtwo-dimensional sugar chain mapping.

(1) Analysis of Neutral Sugar and Amino Sugar Compositions

The sugar chain composition of an antibody composition can be analyzedby carrying out acid hydrolysis of sugar chains with trifluoroaceticacid or the like to release neutral sugars or amino sugars and analyzingthe composition ratio.

Specifically, the analysis can be carried out by a method using acarbohydrate analysis device manufactured by Dionex. BioLC is a devicefor analyzing the sugar composition by HPAEC-PAD (high performanceanion-exchange chromatography-pulsed amperometric detection) [J. Liq.Chromatogr., 6, 1577 (1983)].

The composition ratio can also be analyzed by the fluorescence labelingmethod using 2-aminopyridine. Specifically, the composition ratio can becalculated by fluorescence labeling an acid-hydrolyzed sample by2-aminopyridylation according to a known method [Agric. Biol. Chem.,55(1), 283-284 (1991)] and then analyzing the composition by HPLC.

(2) Analysis of Sugar Chain Structure

The sugar chain structure of an antibody composition can be analyzed bytwo-dimensional sugar chain mapping [Anal. Biochem., 171, 73 (1988);Seibutsukagaku Jikkenho (Biochemical Experimentation Methods)23—Totanpakushitsu Tosa Kenkyuho (Methods of Studies on GlycoproteinSugar Chains), Gakkai Shuppan Center, edited by Reiko Takahashi (1989)].The two-dimensional sugar chain mapping is a method of deducing a sugarchain structure, for example, by plotting the retention time or elutionposition of a sugar chain by reversed phase chromatography as the X axisand the retention time or elution position of the sugar chain by normalphase chromatography as the Y axis, respectively, and comparing themwith the results of known sugar chains.

Specifically, a sugar chain is released from an antibody byhydrazinolysis of the antibody and subjected to fluorescence labelingwith 2-aminopyridine (hereinafter referred to as PA) [J. Biochem., 95,197 (1984)]. After being separated from an excess PA-treating reagent bygel filtration, the sugar chain is subjected to reversed phasechromatography. Then, each peak of the fractionated sugar chain issubjected to normal phase chromatography. The sugar chain structure canbe deduced by plotting the obtained results on a two-dimensional sugarchain map and comparing them with the spots of a sugar chain standard(manufactured by Takara Shuzo Co., Ltd.) or those in the literature[Anal. Biochem., 171, 73 (1988)].

The structure deduced by the two-dimensional sugar chain mapping can beconfirmed by carrying out mass spectrometry, e.g., MALDI-TOF-MS, of eachsugar chain.

5. Method for Determining the Sugar Chain Structure of an AntibodyMolecule

An antibody composition comprises an antibody molecule having differentsugar chain structures binding to the Fc region of antibody. Among theantibody compositions of the present invention, the antibodycomposition, in which the ratio of sugar chains in which fucose is notbound to the N-acetylglucosamine in the reducing terminal to the totalcomplex type N-glycoside-linked sugar chains bound to the Fc region is20% or more, shows high ADCC activity. Such an antibody composition canbe determined using the method for analyzing the sugar chain structureof an antibody molecule described in the above 4. Further, it can alsobe determined by immunoassays using lectins.

Determination of the sugar chain structure of an antibody molecule byimmunoassays using lectins can be made according to the immunoassayssuch as Western staining, RIA (radioimmunoassay), VIA (viroimmunoassay),EIA (enzymeimmunoassay), FIA (fluoroimmunoassay) and MIA(metalloimmunoassay) described in the literature [Monoclonal Antibodies:Principles and Applications, Wiley-Liss, Inc. (1995); EnzymeImmunoassay, 3rd Ed., Igaku Shoin (1987); Enzyme Antibody Technique,Revised Edition, Gakusai Kikaku (1985); and the like], for example, inthe following manner.

A lectin recognizing the sugar chain structure of an antibody moleculeconstituting an antibody composition is labeled, and the labeled lectinis subjected to reaction with a sample antibody composition, followed bymeasurement of the amount of a complex of the labeled lectin with theantibody molecule.

Examples of lectins useful for determining the sugar chain structure ofan antibody molecule include WGA (wheat-germ agglutinin derived from T.vulgaris), ConA (concanavalin A derived from C. ensiformis), RIC (toxinderived from R. communis), L-PHA (leukoagglutinin derived from P.vulgaris), LCA (lentil agglutinin derived from L. culinaris), PSA (pealectin derived from P. sativum), AAL (Aleuria aurantia lectin), ACL(Amaranthus caudatus lectin), BPL (Bauhinia purpurea lectin), DSL(Datura stramonium lectin), DBA (Dolichos biflorus agglutinin), EBL(Elderberry balk lectin), ECL (Erythrina cristagalli lectin), EEL(Euonymus europaeus lectin), GNL (Galanthus nivalis lectin), GSL(Griffonia simplicifolia lectin), HPA (Helix pomatia agglutinin), HHL(Hippeastrum hybrid lectin), Jacalin, LTL (Lotus tetragonolobus lectin),LEL (Lycopersicon esculentum lectin), MAL (Maackia amurensis lectin),MPL (Madura pomifera lectin), NPL (Narcissus pseudonarcissus lectin),PNA (peanut agglutinin), E-PHA (Phaseolus vulgaris erythroagglutinin),PTL (Psophocarpus tetragonolobus lectin), RCA (Ricinus communisagglutinin), STL (Solanum tuberosum lectin), SJA (Sophora japonicaagglutinin), SBA (soybean agglutinin), UEA (Ulex europaeus agglutinin),VVL (Vicia villosa lectin) and WFA (Wisteria floribunda agglutinin)

It is preferred to use lectins specifically recognizing a sugar chainstructure wherein fucose is bound to the N-acetylglucosamine in thereducing terminal in complex type N-glycoside-linked sugar chains.Examples of such lectins include lentil lectin LCA (lentil agglutininderived from Lens culinaris), pea lectin PSA (pea lectin derived fromPisum sativum), broad bean lectin VFA (agglutinin derived from Viciafaba) and Aleuria aurantia lectin AAL (lectin derived from Aleuriaaurantia).

6. Utilization of the Recombinant Antibody Composition of the PresentInvention

Since the recombinant antibody composition of the present invention hashigher CDC activity than an IgG1 antibody and an IgG3 antibody, it hasmore excellent property in therapeutic effects than conventionalantibody compositions. Also, among the antibody compositions of thepresent invention, since the recombinant antibody composition comprisingan antibody molecule having complex type N-glycoside-linked sugar chainsin the Fc region, wherein the ratio of sugar chains in which fucose isnot bound to N-acetylglucosamine in the reducing terminal of the sugarchains among the total complex type N-glycoside-linked sugar chainswhich bind to the Fc region contained in the composition is 20% or morehas higher CDC activity and higher ADCC activity than an IgG1 antibodyand an IgG3 antibody, it has more excellent property in therapeuticeffects than conventional antibody compositions. Furthermore, among therecombinant antibody compositions of the present invention, therecombinant antibody composition comprising an antibody molecule havingcomplex type N-glycoside-linked sugar chains in the Fc region, whereinthe ratio of sugar chains in which fucose is not bound toN-acetylglucosamine in the reducing terminal of the sugar chains amongthe total complex type N-glycoside-linked sugar chains which bind to theFc region contained in the composition is 100% is more preferred.

A medicament comprising the recombinant antibody composition of thepresent invention may be administered alone as a therapeutic agent.However, it is preferably mixed with one or more pharmaceuticallyacceptable carriers and provided as a pharmaceutical preparationproduced by an arbitrary method well known in the technical field ofpharmaceutics.

It is desirable to administer the medicament by the route that is mosteffective for the treatment. Suitable administration routes include oraladministration and parenteral administration such as intraoraladministration, intratracheal administration, intrarectaladministration, subcutaneous administration, intramuscularadministration and intravenous administration. In the case of anantibody preparation, intravenous administration is preferable.

The medicament may be in the form of spray, capsules, tablets, granules,syrup, emulsion, suppository, injection, ointment, tape, and the like.

The preparations suitable for oral administration include emulsions,syrups, capsules, tablets, powders and granules.

Liquid preparations such as emulsions and syrups can be prepared using,as additives, water, sugars (e.g., sucrose, sorbitol and fructose),glycols (e.g., polyethylene glycol and propylene glycol), oils (e.g.,sesame oil, olive oil and soybean oil), antiseptics (e.g.,p-hydroxybenzoates), flavors (e.g., strawberry flavor and peppermint),and the like.

Capsules, tablets, powders, granules, and the like can be preparedusing, as additives, excipients (e.g., lactose, glucose, sucrose andmannitol), disintegrating agents (e.g., starch and sodium alginate),lubricants (e.g., magnesium stearate and talc), binders (e.g., polyvinylalcohol, hydroxypropyl cellulose and gelatin), surfactants (e.g., fattyacid esters), plasticizers (e.g., glycerin), and the like.

The pharmaceutical preparations suitable for parenteral administrationinclude injections, suppositories and sprays.

Injections can be prepared using carriers comprising a salt solution, aglucose solution, or a mixture thereof, etc. It is also possible toprepare powder injections by freeze-drying the antibody compositionaccording to a conventional method and adding sodium chloride thereto.

Suppositories can be prepared using carriers such as cacao butter,hydrogenated fat and carboxylic acid.

The antibody composition may be administered as such in the form ofspray, or sprays may be prepared using carriers which do not stimulatethe oral or airway mucous membrane of a recipient and which can dispersethe antibody composition as fine particles to facilitate absorptionthereof.

Suitable carriers include lactose and glycerin. It is also possible toprepare aerosols, dry powders, and the like according to the propertiesof the antibody composition and the carriers used. In preparing theseparenteral preparations, the above-mentioned additives for the oralpreparations may also be added.

The dose and administration frequency will vary depending on the desiredtherapeutic effect, the administration route, the period of treatment,the patient's age, body weight, and the like. However, an appropriatedose of the active ingredient for an adult person is generally 10 μg/kgto 20 mg/kg per day.

The anti-tumor effect of the antibody composition against various tumorcells can be examined by in vitro tests such as CDC activity measurementand ADCC activity measurement and in vivo tests such as anti-tumorexperiments using tumor systems in experimental animals (e.g., mice).

The CDC activity and ADCC activity measurements and anti-tumorexperiments can be carried out according to the methods described in theliterature [Cancer Immunology Immunotherapy, 36, 373 (1993); CancerResearch, 54, 1511 (1994); and the like].

The present invention is described below based on Examples; however, thepresent invention is not limited thereto.

Example 1 Preparation of Anti-CD20 Human IgG1 Chimeric Antibody,Anti-CD20 Human IgG3 Chimeric Antibody and Anti-CD20 Domain-SwappedAntibody Using Animal Cells 1. Production of Anti-CD20 Human IgG3Chimeric Antibody Expression Vector

cDNA was synthesized from human lymph node-derived poly A+ RNA(manufactured by BD Biosciences Clontech) using cDNA Synthesis Kit(manufactured by Amersham Pharmacia Biotech) in accordance with theinstructions attached thereto. PCR was carried out using 100 ng of cDNAas the template, and using KOD plus (manufactured by TOYOBO) and humanIgG constant region-specific synthetic DNA primers (manufactured byFASMAC) comprising the amino acid sequences represented by SEQ ID NOs:1and 2 in accordance with the attached instructions of KOD plus. PCR wascarried out using GeneAmp™ PCR System 9700 (manufactured by AppliedBiosystems) after thermal denaturation at 94° C. for 1 minute, followedby 30 cycles consisting of reactions at 94° C. for 15 seconds, at 62° C.for 30 seconds and at 68° C. for 90 seconds. After further carrying outreaction at 68° C. for 7 minutes, 2.5 U of Taq DNA polymerase(manufactured by Takara Shuzo) was added thereto and allowed to react at68° C. for 7 minutes in order to add adenine to the 3′-terminal. Thereaction solution was subjected to electrophoresis using 1% agarose gel,and an amplified fragment of about 1.1 kbp considered to be a gene ofthe heavy chain constant region of IgG3 was recovered by using QIAquick™Gel Extraction Kit (manufactured by Qiagen). A ligation reaction with aplasmid pCRII-TOPO vector (manufactured by Invitrogen) was carried outby adding Ligation High solution (manufactured by TOYOBO), andEscherichia coli DH5α (manufactured by TOYOBO) was transformed using thereaction solution. Each plasmid DNA was prepared from the thus obtainedtransformant clones and allowed to react using Big Dye Terminator Cycle™Sequencing Kit v3.1 (manufactured by Applied Biosystems) in accordancewith the instructions attached thereto, and then the nucleotide sequenceof the DNA inserted into the plasmid was analyzed by a DNA sequencer ABIPRISM 3700™ DNA Analyzer of the same company to confirm that thissequence is a nucleotide sequence encoding the same amino acid sequenceof the heavy chain constant region of a conventionally known human IgG3(GenBank accession No. AAH33178).

A gene fragment of 1.13 kbp in the heavy chain constant region of IgG3was purified from the above-described plasmid into which the gene of theheavy chain constant region of human IgG3 was inserted, by treatmentwith restriction enzymes ApaI and NruI (both manufactured by TakaraShuzo). Stable animal cell expression vector for anti-CD20 human IgG1chimeric antibody (described in WO03/055993A1), pKANTEX2B8P, whichcomprises a variable region of an anti-CD20 human IgG1 chimeric antibodyRituxan™, human κ type light chain constant region and human IgG1 heavychain constant region, was digested with ApaI and NruI. Expressionvector for anti-CD20 human IgG3 chimeric antibody, pKANTEX2B8γ3 (FIG. 3)was constructed by cleaving the IgG1 constant region gene, purifying theremaining fragment of about 12.6 kbp and ligating it with theabove-described IgG3 constant region gene fragment using the LigationHigh solution. The amino acid sequences of the variable region and thelight chain constant region of the anti-CD20 human IgG3 chimericantibody encoded by pKANTEX2B8γ3 were identical to the amino acidsequences of the variable region and the light chain constant region ofthe anti-CD20 human IgG1 chimeric antibody encoded by pKANTEX2B8P.

2. Production of Anti-CD20 Domain-Swapped Antibody Expression Vector

A domain-swapped antibody which binds to CD20, wherein the amino acidsequences of the variable region and the light chain constant region areidentical to the amino acid sequences of the variable region and thelight chain constant region of the anti-CD20 human IgG1 chimericantibody encoded by pKANTEX2B8P and the heavy chain constant region isconstituted by the domain of a human IgG1 antibody or human IgG3antibody, was prepared in accordance with the following procedure. Theanti-CD20 chimeric antibody having a heavy chain constant region inwhich the CH1 and hinge are constituted by amino acid sequences from ahuman IgG1 antibody, and the Fc regions (CH2 and CH3) are constituted byamino acid sequences from a human IgG3 antibody, is called 1133-typeanti-CD20 domain-swapped antibody, and the anti-CD20 chimeric antibodyhaving a heavy chain constant region wherein the CH1 and hinge areconstituted by amino acid sequences from a human IgG3 antibody, and theFc regions are constituted by amino acid sequences from a human IgG1antibody, is called 3311-type anti-CD20 domain-swapped antibody. As aresult of search using amino acid sequence database, it was found thatthe amino acid sequences of heavy chain constant regions of thesedomain-swapped antibodies are novel amino acid sequences.

Subclasses from which each domain of the various designed anti-CD20domain-swapped antibodies was derived, and corresponding amino acidsequences of heavy chain constant regions are shown in Table 1.Schematic illustration of each anti-CD20 domain-swapped antibody isshown in FIG. 4.

TABLE 1 Fc Structural name CH1 Hinge CH2 CH3 Amino acid sequence 1133IgG1 IgG1 IgG3 IgG3 SEQ ID NO: 16 3311 IgG3 IgG3 IgG1 IgG1 SEQ ID NO: 4

(1) Construction of Expression Vector Encoding the 1133-Type Anti-CD20Domain-Swapped Antibody

The expression vector encoding the 1133-type anti-CD20 domain-swappedantibody, shown in FIG. 5, was constructed in the following manner.

A DNA fragment of about 430 bp encoding CH1 domain, hinge domain and apart of the 5′-terminal side of Fc region (a part in which the aminoacid sequence was identical between human IgG1 antibody and human IgG3antibody) of the human IgG1 antibody was cleaved and purified from theexpression vector for anti-CD20 human IgG1 chimeric antibody,pKANTEX2B8P using restriction enzymes ApaI (manufactured by TakaraShuzo) and BmgBI (manufactured by New England Biolabs). On the otherhand, a DNA fragment of about 13 kbp was cleaved and purified from theexpression vector for anti-CD20 human IgG3 chimeric antibody,pKANTEX2B8γ3 described in the item 1 of this Example by the similartreatment with restriction enzymes. After mixing these purified DNApreparations, a ligation reaction was carried out using Ligation Highsolution (manufactured by TOYOBO), and Escherichia coli XL1-BLUE MRF′(manufactured by Stratagene) was transformed using the reactionsolution. Each plasmid DNA was prepared from the thus obtainedtransformant clones and allowed to react using Big Dye Terminator Cycle™Sequencing Kit v3.1 (manufactured by Applied Biosystems) in accordancewith the instructions attached thereto, and then the nucleotide sequenceof the DNA inserted into the plasmid was analyzed by a DNA sequencer ABIPRISM 3700™ DNA Analyzer of the same company to confirm that the plasmidpKTX93/1133 shown in FIG. 5 was obtained.

(2) Construction of Expression Vector Encoding the 3311-Type Anti-CD20Domain-Swapped Antibody

The expression vector encoding the 3311-type anti-CD20 domain-swappedantibody, shown in FIG. 6, was constructed in the following manner.

A DNA fragment of about 570 bp encoding CH1 domain, hinge domain and apart of the 5′-terminal side of Fc region (a part in which the aminoacid sequence was identical between human IgG1 antibody and human IgG3antibody) of the human IgG3 antibody was cleaved and purified from thehuman IgG3 chimeric antibody expression vector, pKANTEX2B8γ3 describedin the item 1 of this Example using restriction enzymes ApaI(manufactured by Takara Shuzo) and BmgBI (manufactured by New EnglandBiolabs). On the other hand, a DNA fragment of about 13 kbp was cleavedand purified from the expression vector for IgG1 anti-CD20 antibody,pKANTEX2B8P by the similar treatment with restriction enzymes. Aftermixing these purified DNA preparations, a ligation reaction was carriedout using Ligation High solution (manufactured by TOYOBO), andEscherichia coli XL1-BLUE MRF′ (manufactured by Stratagene) wastransformed using the reaction solution. Each plasmid DNA was preparedfrom the thus obtained transformant clones and allowed to react usingBig Dye Terminator Cycle™ Sequencing Kit v3.1 (manufactured by AppliedBiosystems) in accordance with the instructions attached thereto, andthen the nucleotide sequence of the DNA inserted into the plasmid wasanalyzed by a DNA sequencer ABI PRISM 3700™ DNA Analyzer of the samecompany to confirm that the plasmid pKTX93/3311 shown in FIG. 6 wasobtained.

3. Stable Expression of Various Anti-CD20 Chimeric Antibodies andVarious Anti-CD20 Domain-Swapped Antibodies in Animal Cells

Cells for stably producing an anti-CD20 human IgG3 chimeric antibody oranti-CD20 domain-swapped antibody, in which the expression vector foranti-CD20 human IgG3 chimeric antibody, pKANTEX2B8γ3 and expressionvectors for anti-CD20 domain-swapped antibody, pKTX93/1133 andpKTX93/3311 prepared in the items 1 and 2 of this Example, wereintroduced into a CHO/DG44 cell [Somatic Cell Mol. Genet., 12, 555(1986)] and the CHO/DG44 cell in which α1,6-fucosyltransferase gene wasknocked out (hereinafter referred to as CHO/FUT8^(−/−)) [Biotechnol.Bioeng., 87, 614 (2004)] as host cells were prepared in the followingmanner. The CHO/DG44 cell is a host cell widely used in the productionof recombinant protein. The CHO/FUT8^(−/−) is a host cell in which FUT8of the CHO/DG44 cell is knocked out on the genome. In addition, theexpression vector pKANTEX2B8P for anti-CD20 human IgG1 chimeric antibodywas introduced into the CHO/FUT8^(−/−) cell alone, and a cell capable ofstably producing an anti-CD20 human IgG1 chimeric antibody was preparedin the same manner.

After introducing 8 μg of each expression vector plasmid into 1.6×10⁶cells of the CHO/DG44 cell or CHO/FUT8^(−/−) cell by the electroporationmethod [Cytotechnology, 3, 133 (1990)], the cells were suspended in 40ml of IMDM-(10) [IMDM medium (manufactured by GIBCO-BRL) containing 10%of dialyzed fetal bovine serum (dFBS)] and dispensed at 100 μl/well intoa 96-well microplate (manufactured by Sumitomo Bakelite). Afterculturing at 37° C. for 24 hours in a 5% CO₂ incubator, the cells werecultured for 1 to 2 weeks in the IMDM-(10) containing G418 atconcentration of 500 μg/ml. After the culturing, culture supernatant wasrecovered from each well, and the amount of the anti-CD20 domain-swappedantibody in the culture supernatant was measured by the ELISA which isdescribed later in the item 4 of this Example. Regarding thetransformants of wells in which expression of the anti-CD20domain-swapped antibody was found in the culture supernatants, in orderto increase the antibody expression amount using the dhfr geneamplification system, the cells were suspended in the IMDM-(10) mediumcontaining G418 at concentration of 500 μg/ml and methotrexate atconcentration of 50 nM (hereinafter referred to as MTX: manufactured bySIGMA) as an inhibitor of dihydrofolate reductase which was the dhfrgene product and cultured at 37° C. for about 1 week in a 5% CO₂incubator to thereby obtain transformants having resistance to 50 nM ofMTX. Subsequently, the MTX concentration was successively raised to 100nM and then to 200 nM to finally obtain transformants which canproliferate in the IMDM-(10) medium containing G418 at concentration of500 μg/ml and 200 nM MTX and also can express the antibodies encoded bythe respective expression vectors at high level.

4. Measurement of Antibody Concentration in Culture Supernatant (ELISA)

Goat anti-human IgG (H & L) antibody (manufactured by American Qualex)was diluted to 1 μg/ml with phosphate buffered saline (hereinafterreferred to as PBS; manufactured by Proliant Inc), dispensed at 50μl/well into a 96-well plate for ELISA (manufactured by Greiner) andallowed to stand at room temperature for 1 hour for adsorption. Afterthe reaction, the plate was washed with PBS, and 1% bovine serum albumin(hereinafter referred to as BSA)-containing PBS (hereinafter referred toas 1% BSA-PBS) was added thereto at 100 μl/well and allowed to react atroom temperature for 1 hour to block the remaining active groups. Afterremoving 1% BSA-PBS, culture supernatants to be measured were added at50 μl/well and allowed to react at room temperature for 2 hours. Afterthe reaction, each well was washed with 0.05% Tween 20-containing PBS(hereinafter referred to as Tween-PBS), and then a peroxidase-labeledgoat anti-human IgG (Fc) antibody solution (manufactured by AmericanQualex) diluted 500-fold with PBS was added at 50 μl/well as thesecondary antibody solution and allowed to react at room temperature for1 hour. After washing with Tween-PBS, ABTS substrate solution [asolution prepared by dissolving 0.55 g of ammonium2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) in 1 liter of 0.1 Mcitrate buffer (pH 4.2) and adding 1 μl/ml of hydrogen peroxide justbefore the use] was added at 50 μl/well for color development, and theabsorbance at 415 nm (hereinafter referred to as OD415) was measured.

5. Purification of Various Anti-CD20 Chimeric Antibodies and VariousAnti-CD20 Domain-Swapped Antibodies

Each of the transformants capable of expressing various anti-CD20antibodies obtained in the item 3 of this Example was suspended inIMDM-FCS(10) containing 200 nM of MTX to a density of 1×10⁵ cells/ml,and then dispensed at 100 ml into triple flasks (manufactured byNalgenunc) and cultured at 37° C. for 2 days in a 5% CO₂ incubator.Culture supernatant was removed from each flask, the inside of the flaskwas washed with 50 ml of PBS, and then 100 ml of EXCELL 301 medium(manufactured by JRH Biosciences) was added to the flask to continue theculturing at 37° C. for 5 days in the 5% CO₂ incubator. This culturesupernatant was recovered, centrifuged at 3000 rpm and 4° C. for 5minutes, and then the supernatant was recovered and subjected tofiltration sterilization using a 0.22 μm PES Membrane (manufactured byIwaki). The various anti-CD20 antibodies were purified from the thussterilized culture supernatants using a column packed with Prosep-A™(Protein-A: manufactured by Millipore) or Prosep-G™ (Protein-G:manufactured by Millipore) in accordance with the instructions attachedthereto. The IgG1 anti-CD20 antibody was purified by protein A, butsince the IgG3 anti-CD20 antibody was not purified by protein A,purification was carried out by using protein G. Regarding thedomain-swapped antibodies, the 3311-type was purified by protein A. Onthe other hand, the 1133-type was purified with protein A, but could bepurified by protein G.

The expression vector and host cell of each antibody, names of thepurified antibody samples and corresponding heavy chain constant regionof amino acid sequences are shown in Table 2. In this connection, in thetable, the sample having(+F) at the end of the sample name indicates anantibody sample produced using CHO/DG44 as the host cell, and othersamples indicate antibody samples produced from CHO/FUT8^(−/−).

TABLE 2 Expression vector Host cell Purified antibody (name) pKANTEX2B8CHO/FUT8^(−/−) CD20-IgG1(−F) pKANTEX2B8g3 CHO/DG44 CD20-IgG3(+F)pKANTEX2B8g3 CHO/FUT8^(−/−) CD20-IgG3(−F) pKTX93/1133 CHO/DG44 1133(+F)pKTX93/1133 CHO/FUT8^(−/−) 1133(−F) pKTX93/3311 CHO/DG44 3311(+F)pKTX93/3311 CHO/FUT8^(−/−) 3311(−F) In the table, +F indicates thatfucose is bound to a sugar chain which binds to the Fc region, and −Findicates that fucose is not bound to a sugar chain which binds to theFc region.

6. Evaluation of the Purification Degree of Various Anti-CD20 ChimericAntibody Samples and Various Anti-CD20 Domain-Swapped Antibody SamplesPurified by SDS-PAGE

In order to evaluate the purification degree of the purified samples ofvarious anti-CD20 antibodies obtained in the item 5 of this Example,SDS-polyacrylamide gel electrophoresis (hereinafter referred to asSDS-PAGE) was carried out in accordance with a conventionally knownmethod [Nature, 227, 680 (1970)], using about 1 μg of each of thepurified samples of various anti-CD20 antibodies. As a comparativecontrol of the electrophoresis degree, the same operation was alsocarried out for an anti-CD20 human IgG1 chimeric antibody Rituxan™(purchased from Genentech). Hereinafter, Rituxan™ is referred to asCD20-IgG1(+F).

As a result, 1133(+F) and 1133(−F) showed an electrophoresis patternsimilar to that of the human IgG1 antibody CD20-IgG1(+F), and 3311(+F)and 3311(−F) showed an electrophoresis pattern similar to that of thehuman IgG3 antibody CD20-IgG3(+F). In the case of CD20-IgG1(+F),CD20-IgG1(−F), 1133(+F) and 1133(−F), the band of the H chain was foundat about 50 kilodaltons (hereinafter referred to as kDa), and that ofthe L chain was found at about 24 kDa, and in the case of CD20-IgG3(+F),CD20-IgG3(−F), 3311(+F) and 3311(−F), the band of the H chain was foundat about 54 kDa, and that of the L chain was found at about 24 kDa, sothat it was confirmed that each of the prepared anti-CD20 antibodies isconstituted by the desired H chain and L chain.

Based on the above results, it was confirmed that the various desiredIgG molecules constituted by H chain and L chain are contained at asufficient ratio in the purified samples of respective anti-CD20antibodies obtained in the item 5 of this Example.

Example 2 Activity Evaluation of Various Anti-CD20 Chimeric Antibodiesand Various Anti-CD20 Domain-Swapped Antibodies:

Comparison of various activities was carried out for the purifiedsamples of various anti-CD20 antibodies obtained in the item 5 ofExample 1 in the following manner.

1. Binding Activity of Various Anti-CD20 Antibodies to CD20-PositiveCell

Binding activity of the various anti-CD20 antibodies obtained in Example1 to CD20-positive cells was measured in a competitive inhibition systemwith biotinylated Rituxan™, by fluorescent antibody technique using aflow cytometer. As negative controls, an anti-Her2 human IgG1 antibodyHerceptin™ [Proc. Natl. Acad. Sci. U.S.A., 89, 4285 (1992)] (purchasedfrom Genentech) and an anti-CCR4 human IgG1 antibody KM3060 [CancerRes., 64, 2127 (2004)] were used.

A CD20-positive Burkitt lymphoma-derived cell line Daudi cell (ATCC:CCL-213) was dispensed at 5×10⁵ cells per well into a 96-well U-plate(manufactured by Falcon), and then a buffer for FACS [0.2 mg/ml humanIgG (manufactured by Sigma), 0.02% EDTA, 0.05% NaN₃, 1% BSA] containing10 μg/ml or 1 μg/ml of the respective CD20 antibodies obtained in theitem 5 of Example 1, or the negative controls anti-Her2 antibodyHerceptin™ [Proc. Nall. Acad. Sci. U.S.A., 89, 4285 (1992)] andanti-CCR4 antibody KM3060 (WO02/31140), and containing 0.5 μg/ml ofbiotin-labeled anti-CD20 chimeric antibody Rituxan™ [prepared bybiotinylating Rituxan™ using EZ-Link™ Sulfo-NHS-LC-Biotin (manufacturedby Pierce)], was added thereto at 50 μl/well. After reaction at 4° C.for 60 minutes under shade, the cells were washed twice with the bufferfor FACS, and then the PE-labeled streptoavidin diluted 200-fold withthe buffer for FACS was added thereto at 50 μl/well. After reaction at4° C. for 60 minutes under shade, the cells were washed twice with thebuffer for FACS and suspended in 1 ml of the buffer for FACS, and thenthe fluorescence intensity was measured with a flow cytometer EPICS-XL™(manufactured by Coulter).

The results are shown in FIG. 7. The negative controls anti-Her2antibody Herceptin™ and anti-CCR4 antibody KM3060 did not inhibitbinding of the biotin-labeled anti-CD20 chimeric antibody Rituxan™ tothe CD20-positive cell Daudi, but all of the anti-CD20 domain-swappedantibodies, anti-CD20 human IgG1 chimeric antibodies and anti-CD20 humanIgG3 chimeric antibodies concentration dependently inhibited the bindingand the degree was almost the same. Based on these results, it was shownthat antigen-binding of the anti-CD20 domain-swapped antibodies isCD20-specific and that the binding activity of the anti-CD20domain-swapped antibodies is similar to that of the anti-CD20 human IgG1chimeric antibody.

2. Measurement of CDC Activity of Various Anti-CD20 Antibodies to DaudiCell

In vitro CDC activity of the purified samples of various anti-CD20antibodies obtained in the item 5 of Example 1 was measured using aCD20-positive Daudi cell.

The reaction was carried out in a 96-well flat-bottomed plate(manufactured by Sumitomo Bakelite), and a human complement dilutionmedium [prepared by diluting a human complement (manufactured by SIGMA)6-fold with RPMI 1640 medium (manufactured by GIBCO BRL) containing 10%FBS (manufactured by JRH)] containing 5×10⁴ cells of the Daudi cell andcontaining 0.3 μg/ml of each anti-CD20 domain-swapped antibody,anti-CD20 human IgG1 chimeric antibody or anti-CD20 human IgG3 chimericantibody was dispensed at 150 μl into respective reaction wells. Inaddition, a reaction well containing no anti-CD20 domain-swappedantibody (0% reaction well) was prepared as a control in case CDC wasnot induced, and a reaction well containing no Daudi cell (100% reactionwell) as a control in case CDC was induced. After culturing at 37° C.for 2 hours in an atmosphere of 5% CO₂, WST-1 reagent (manufactured byROCHE) was added at 15 μl into respective reaction wells and allowed toreact at 37° C. for 4 hours in an atmosphere of 5% CO₂. After completionof the reaction, OD450 in each well was measured, and the CDC activity(%) was calculated from the absorbance of each well using the followingformula:

CDC activity (%)=100×{1−(reaction well absorbance−100% reaction wellabsorbance)/(0% reaction well absorbance−100% reaction well absorbance)}

The results are shown in FIG. 8. As shown in FIG. 8, the CDC activity ofanti-CD20 human IgG3 chimeric antibodies CD20-IgG3(+F) and CD20-IgG3(−F)was higher than that of anti-CD20 human IgG1 chimeric antibodiesCD20-IgG1(+F) and CD20-IgG1(−F), so that it was confirmed that the CDCactivity of IgG3 is higher than that of IgG1. However, 1133(+F)-type and1133(−F)-type anti-CD20 domain-swapped antibodies showed considerablyhigher CDC activity than the CDC activity of anti-CD20 human IgG3chimeric antibodies. On the other hand, the CDC activity of anti-CD20domain-swapped antibodies 3311(+F) and 3311(−F) was low. Also, in all ofthe anti-CD20 antibodies, the antibody samples produced using CHO/DG44as the host cell and the antibody samples produced using CHO/FUT8^(−/−)as the host cell showed almost the same CDC activity, and the activityof 1133-type was increased regardless of the fucose content of the sugarchain binding to the antibody. In addition, tendency of the amount ofCDC activity of the above-described various antibodies did not changewhen the antibody concentration was increased to 1 μg/ml.

3. CDC Activity Measurement of 1133-Type Anti-CD20 Domain-SwappedAntibodies

In order to further fully evaluate CDC activity of the 1133(+F)-type and1133(−F)-type anti-CD20 domain-swapped antibodies which showedparticularly high CDC activity in the item 2 of this Example,measurement of CDC activity was carried out in the same manner as in theitem 2 of this Example using a CD20-positive Burkitt lymphoma-derivedcell line ST 486 cell (ATCC: CRL-1647) or Burkitt lymphoma-derived cellline Raji cell (ATCC: CCL-86).

The results are shown in FIG. 9. As shown in FIG. 9, in each of the ST486 cell line (FIG. 9A) and Raji cell line (FIG. 9B), the CDC activityof anti-CD20 human IgG3 chimeric antibodies CD20-IgG3(+F) andCD20-IgG3(−F) was slightly higher than the CDC activity of anti-CD20human IgG1 chimeric antibodies CD20-IgG1(+F) and CD20-IgG1(−F), and1133(+F)-type and 1133(−F)-type anti-CD20 domain-swapped antibodiesshowed remarkable CDC activity exceeding them. In addition, in all ofthese anti-CD20 antibodies, the antibody samples produced by CHO/DG44 asthe host cell and the antibody samples produced by CHO/FUT8^(−/−) as thehost cell showed almost the same CDC activity.

5. Evaluation of ADCC Activity of Various Anti-CD20 Antibodies toCD20-Positive Cell Line

In vitro ADCC activity of the purified samples of various anti-CD20antibodies obtained in the item 5 of Example 1 was measured in thefollowing manner using a CD20-positive Daudi cell as the target cell.Cytotox 96™ Kit (manufactured by Promega) was used in the measurement.

(1) Preparation of Human Effector Cell Suspension

From a healthy volunteer, 50 ml of peripheral blood was collected andgently mixed with 0.2 ml of heparin sodium (manufactured by TakedaPharmaceutical). A monocyte fraction was separated from this usingLymphoprep (manufactured by Daiichi Pure Chemicals) in accordance withthe instructions attached thereto and then washed by centrifugation oncewith RPMI 1640 medium and once with 10% FBS-RPMI 1640 medium, and thecell was used as the effector cell.

(2) Measurement of ADCC Activity

The reaction was carried out in a 96-well flat-bottomed plate(manufactured by Falcon), and 10% FBS-RPMI 1640 medium containing 2×10⁵cells of the effector cell and 1×10⁴ cells of the Daudi cell or ST 486cell and containing each CD20 antibody at varied concentration wasdispensed at 200 μl into each reaction well. In addition, a medium wellwithout the effector cell, target cell and antibody, an effector wellcontaining the effector cell alone, a target well containing the targetcell alone, an NK well containing the effector cell and target cellwithout antibody, a 100% reaction well containing the target cell aloneand to which 20 μl of the Lysis buffer attached to the kit was added 3hours and 15 minutes after commencement of the reaction, and a 100%reaction control well without the effector cell, target cell andantibody and to which 20 μl of the Lysis buffer attached to the kit wasadded 3 hours and 15 minutes after commencement of the reactions, wererespectively prepared as subjective wells necessary for calculating ADCCactivity. After carrying out reaction at 37° C. for 4 hours under anatmosphere of 5% CO₂ in each reaction well, the reaction plate wascentrifuged to recover 50 μl of supernatant from each well. Thesupernatants of wells were respectively transferred to the wells of a96-well U-bottom plate (manufactured by Sumitomo Bakelite), and acoloring substrate solution (prepared by dissolving one ampoule of thesubstrate attached to the kit in 12 ml of the assay buffer attached tothe kit) was added at 50 μl into each well. The coloring reaction wascarried out at 37° C. for 30 minutes, the reaction termination solutionattached to the kit was added at 50 μl to each well, and then OD450 wasmeasured to calculate the ADCC activity (%) from the absorbance of eachwell using the following formula.

ADCC activity (%)=100×(S−E−T)/(Max−T)

-   -   S=sample reaction well absorbance−medium well absorbance    -   E=effector well absorbance−medium well absorbance    -   T=target well absorbance−medium well absorbance    -   Max=100% reaction well−100% reaction control well

The results are shown in FIG. 10. As shown in FIG. 10, in all of theanti-CD20 antibodies, the antibody samples produced from CHO/FUT8^(−/−)showed higher ADCC activity than the antibody samples produced fromCHO/DG44. From this result, it was found that, also in the case of allof the anti-CD20 domain-swapped antibodies prepared in this Example, theADCC activity is increased by the antibody composition in which fucoseis not bound to the N-acetylglucosamine existing in the reducingterminal in the complex type N-glycoside-linked sugar chain bound to theFc of the antibody, in comparison with the antibody composition in whichfucose is bound to the N-acetylglucosamine existing in the reducingterminal of the complex type N-glycoside-linked sugar chain bound to theFc of the antibody. Also, it was confirmed that the anti-CD20 human IgG1chimeric antibodies show higher ADCC activity than that of the anti-CD20human IgG3 chimeric antibodies, that is, ADCC activity of IgG is higherthan that of IgG1. Also, the 1133-type anti-CD20 domain-swappedantibodies maintained high ADCC activity similar to the level ofanti-CD20 human IgG1 chimeric antibodies. In addition, it was found thatADCC activity of the 3311-type anti-CD20 domain-swapped antibodies islow similarly to the case of anti-CD20 human IgG3 chimeric antibodies.

5. Measurement of the Binding Activity of Various Anti-CD20 Antibodiesto Recombinant Fcγ Receptor IIIa (Hereinafter Referred to as FcγRIIIa)

In order to analyze the ADCC activity enhancing mechanism by anti-CD20domain-swapped antibodies confirmed in the item 4 of this Example, thebinding activity of anti-CD20 human IgG1 chimeric antibodiesCD20-IgG1(−F) and CD20-IgG1(+F), anti-CD20 human IgG3 chimericantibodies CD20-IgG3(−F) and CD20-IgG3(+F), and 1133-type anti-CD20domain-swapped antibodies 1133(−F) and 1133(+F) to an Fc receptor familyFcγRIIIa expressing on the surface of NK cell was measured in accordancewith a conventionally known method [Clin. Cancer Res., 10, 6248 (2004)].

The results are shown in FIG. 11. As shown in FIG. 11, the antibodysamples produced by CHO/FUT8^(−/−) showed higher binding activity forFcγRIIIa than that of the antibody samples produced by CHO/DG44. Basedon this result, it was confirmed that the increase of ADCC activity ofantibody, due to the removal of the fucose binding to theN-acetylglucosamine existing in the reducing terminal of the complextype N-glycoside-linked sugar chain which is added to the Fc of1133-type anti-CD20 domain-swapped antibodies, is caused by increasingthe activity of the Fc region to the Fc receptor.

Based on the above, the 1133-type anti-CD20 domain-swapped antibodieshaving the same variable region as the anti-CD20 human IgG1 chimericantibody Rituxan™, in which the CH1 domain and hinge domain of the Hchain are the amino acid sequences of human IgG1 antibody and the Fcregion is those of human IgG3 antibody, have CDC activity that exceedsanti-CD20 human IgG1 chimeric antibodies and anti-CD20 human IgG3chimeric antibodies and also have ADCC activity substantially equivalentto that of the anti-CD20 human IgG1 chimeric antibodies. In addition, itwas shown that the activity of binding Fc to Fc receptor is increasedand the ADCC activity is improved similarly to the case of the anti-CD20human IgG1 chimeric antibodies, by decreasing the content of fucosebinding to the N-acetylglucosamine existing in the reducing terminal inthe complex type N-glycoside-linked sugar chain bound to the Fc.

Relationship between structures and activities of each of the preparedrespective antibody and domain-swapped antibodies is summarized in Table3 based on the results obtained in the above. In the table, ADCCactivity and CDC activity were expressed as ++++, +++, ++ and + in orderof the grade of activities.

TABLE 3 Purified antibody ADCC CDC (name) CH1 Hinge CH2 CH3 activityactivity CD20-IgG1(+F)/ IgG1 IgG1 IgG1 IgG1 ++/+++ ++ CD20-IgG1(−F)CD20-IgG3(+F)/ IgG3 IgG3 IgG3 IgG3 +/++ +++ CD20-IgG3(−F)1133(+F)/1133(−F) IgG1 IgG1 IgG3 IgG3 ++/++ ++++ 3311(+F)/3311(−F) IgG3IgG3 IgG1 IgG1 +/+ ++

Based on the above, it was shown that an antibody molecule having aheavy chain constant region in which the Fc region of the human IgG1antibody was replaced by the Fc region of the human IgG3 antibody hasCDC activity higher than that of the human IgG1 antibody and human IgG3antibody and maintains high ADCC activity substantially equivalent tothat of the human IgG1 antibody.

Example 3 Production of 1131-Type Anti-CD20 Domain-Swapped Antibody and1113-Type Anti-CD20 Domain-Swapped Antibody Using Animal Cell: 1.Production of Expression Vector for 1131-Type Anti-CD20 Domain-SwappedAntibody and Expression Vector for 1113-Type Anti-CD20 Domain-SwappedAntibody

In Example 2, the 1133-type anti-CD20 domain-swapped antibody preparedby replacing the Fc region (CH2 and CH3) of the anti-CD20 human IgG1chimeric antibody with the Fc region of the human IgG antibody showedCDC activity higher than that of anti-CD20 human IgG1 chimeric antibody.Next, in order to individually examine participation of the CH2 domainand CH3 domain which constitute the Fc region in the CDC activity, thefollowing two anti-CD20 domain-swapped antibodies were prepared.

In the following Example, an anti-CD20 chimeric antibody having a heavychain constant region in which the CH1, hinge and CH3 are constituted bythe amino acid sequences from a human IgG1 antibody, and the CH2 isconstituted by the amino acid sequences from a human IgG3 antibody, iscalled 1131-type anti-CD20 domain-swapped antibody, and an anti-CD20chimeric antibody having a heavy chain constant region in which the CH1,hinge and CH2 are constituted by the amino acid sequences from a humanIgG1 antibody, and the CH3 domain constituted by the amino acidsequences from a human IgG3 antibody, is called 1113-type anti-CD20domain-swapped antibody. In each case, amino acid sequences of thevariable region and light chain constant region are identical to theamino acid sequences of the variable region and light chain constantregion of the anti-CD20 human IgG1 chimeric antibody encoded bypKANTEX2B8P.

Domain structures and amino acid sequences of the heavy chain constantregions of the 1131-type anti-CD20 domain-swapped antibody and 1113-typeanti-CD20 domain-swapped antibody are shown in Table 4. Since noexamples for preparing heavy chain constant regions of these anti-CD20domain-swapped antibodies are unknown, both of them are novelstructures. In addition, a schematic illustration of each domain-swappedantibody is shown in FIG. 12.

TABLE 4 Structure name CH1 Hinge CH2 CH3 Amino acid sequence 1113-typeIgG1 IgG1 IgG1 IgG3 SEQ ID NO: 6 1131-type IgG1 IgG1 IgG3 IgG1 SEQ IDNO: 31

(1) Construction of Expression Vector Comprising Nucleotide SequenceEncoding 1113-Type Anti-CD20 Domain-Swapped Antibody

An expression vector encoding the 1113-type anti-CD20 chimeric antibody,wherein the amino acid sequences of the variable region and light chainconstant region are identical to the amino acid sequences of thevariable region and light chain constant region of the anti-CD20 humanIgG1 chimeric antibody encoded by pKANTEX2B8P, and it has a heavy chainconstant region wherein the CH1, hinge and CH2 are constituted by theamino acid sequences from a human IgG3 antibody, and the CH3 domain isconstituted by the amino acid sequences from a human IgG1 antibody, wasconstructed in the following manner.

A DNA fragment of about 700 bp encoding the human IgG1 CH1 domain, hingedomain and CH2 domain was cleaved and purified from the expressionvector for anti-CD20 human IgG1 chimeric antibody, pKANTEX2B8P shown inFIG. 13, using restriction enzymes ApaI (manufactured by Takara Shuzo)and SmaI (manufactured by Takara Shuzo). On the other hand, a DNAfragment of about 13 kbp was cleaved and purified by the same treatmentwith restriction enzymes on the expression vector for 1133-typeanti-CD20 domain-swapped antibody, pKANTEX93/1133 described in the item2(2) of Example 1 and shown in FIG. 14. After mixing these purified DNApreparations, a ligation reaction was carried out using Ligation Highsolution (manufactured by TOYOBO), and Escherichia coli XL1-BLUE MRF′(manufactured by Stratagene) was transformed using the reactionsolution. Each plasmid DNA was prepared from the thus obtainedtransformant clones and allowed to react using Big Dye Terminator Cycle™Sequencing Kit v3.1 (manufactured by Applied Biosystems) in accordancewith the instructions attached thereto, and then the nucleotide sequenceof the DNA inserted into each plasmid was analyzed by a DNA sequencerABI PRISM 3700™ DNA Analyzer of the same company to confirm that theplasmid pKTX93/1113 shown in FIG. 15 was obtained.

(2) Construction of Expression Vector Comprising Nucleotide SequenceEncoding the Gene of 1131-Type Anti-CD20 Domain-Swapped Antibody 1131

An expression vector encoding the 1113-type domain-swapped antibodyshown in FIG. 16 which specifically reacts with human CD20, wherein theCH2 domain of CH is the amino acid sequence of human IgG3 and the CH1domain, hinge domain and CH3 domain are the amino acid sequences ofhuman IgG1, was constructed in the following manner.

A DNA fragment of about 700 bp encoding the human IgG1 CH1 domain andhinge domain and the human IgG3 CH2 domain was cleaved and purified fromthe expression vector for 1133-type anti-CD20 domain-swapped antibody,pKANTEX93/1133 described in the item 2(2) of Example 1 and shown in FIG.14, using restriction enzymes ApaI (manufactured by Takara Shuzo) andSmaI (manufactured by Takara Shuzo). On the other hand, a DNA fragmentof about 13 kbp was cleaved and purified by carrying out the samerestriction enzyme treatment on the expression vector for anti-CD20human IgG1 chimeric antibody, pKANTEX2B8P shown in FIG. 13. After mixingthese purified DNA preparations, a ligation reaction was carried outusing Ligation High solution (manufactured by TOYOBO), and Escherichiacoli XL1-BLUE MRF′ (manufactured by Stratagene) was transformed usingthe reaction solution. Each plasmid DNA was prepared from the thusobtained transformant clones and allowed to react using Big DyeTerminator Cycle™ Sequencing Kit v3.1 (manufactured by AppliedBiosystems) in accordance with the instructions attached thereto, andthen the nucleotide sequence of the DNA inserted into each plasmid wasanalyzed by a DNA sequencer ABI PRISM 3700™ DNA Analyzer of the samecompany to confirm that the plasmid pKTX93/1131 shown in FIG. 16 wasobtained.

2. Stable Expression of 1113-Type and 1131-Type Anti-CD20 Domain-SwappedAntibodies in Animal Cell

A cell which stably produces the anti-CD20 antibody domain-swappedantibody was prepared in the same manner as in the item 3 of Example 1.The expression vector for anti-CD20 domain-swapped antibody prepared inthe item 1 of this Example was introduced into the CHO/FUT8^(−/−)described in the item 3 of Example 1 as the host cell.

3. Purification of Anti-CD20 Domain-Swapped Antibody

The transformant obtained in the item 2 of this Example capable ofexpressing the 1113-type anti-CD20 domain-swapped antibody or 1131-typeanti-CD20 domain-swapped antibody was cultured and purified in the samemanner as in the item 5 of Example 1. The 1113-type anti-CD20domain-swapped antibody and 1131-type anti-CD20 domain-swapped antibodywere purified using the Prosep-G column. In addition, when the 1133-typeanti-CD20 domain-swapped antibody, 1113-type anti-CD20 domain-swappedantibody and 1131-type anti-CD20 domain-swapped antibody were purifiedusing Prosep-A column, only the 1131-type anti-CD20 domain-swappedantibody was capable of being purified.

The expression vector and host cell of each domain-swapped antibody andname of the purified antibody are shown in Table 5.

TABLE 5 Expression vector Host cell Purified antibody (name) pKTX93/1113CHO/FUT8^(−/−) 1113(−F) pKTX93/1131 CHO/FUT8^(−/−) 1131(−F)

4. Evaluation of Purification Degree of Purified Anti-CD20Domain-Swapped Antibodies by SDS-PAGE

In order to measure purification degree of the purified samples ofvarious anti-CD20 domain-swapped antibodies obtained in the item 3 ofthis Example, SDS-PAGE was carried out in the same manner as in the item6 of Example 1. As comparative controls of electrophoresis, the sameoperation was also carried out for the respective purified samples ofCD20-IgG1-type, CD20-IgG3-type and 1133-type prepared in the item 5 ofExample 1.

The results are shown in FIG. 17. The 1113-type and 1131-type showedelectrophoresis patterns similar to the CD20-IgG1-type and 1133-type,respectively. The molecular weights deduced from the amino acidsequences of H chain and L chain constituting the 1113-type and1131-type are similar to each other, and the H chain is about 50 kDa andthe L chain is about 24 kDa. Since these molecular weights are similarto the H chain and L chain molecular weights of the CD20-IgG1-type and1133-type, and the electrophoresis patterns are also similar thereto, itwas confirmed that the 1113-type and 1131-type are constituted by thedesired H chain and L chain. In addition, the molecular weight deducedfrom the amino acid sequence of L chain constituting the CD20-IgG3-typewas about 24 kDa which is similar to that of the CD20-IgG1-type, but theH chain constituting the CD20-IgG3-type was about 54 kDa which is largerthan that of the H chain of the CD20-IgG1-type, so that L chain of theCD20-IgG3-type appeared at a position similar to that of the L chain ofthe CD20-IgG1-type, but the bond of H chain of the CD20-IgG3-type waspositioned at a high molecular weight side than that of H chain of theCD20-IgG1-type.

From the above results, it was confirmed that the desired IgG moleculesrespectively constituted by H chain and L chain are contained at asufficient ratio in the purified samples of various anti-CD20domain-swapped antibodies obtained in the item 3 of this Example.

Example 4 Activity Evaluation of 1131-Type and 1113-Type Anti-CD20Domain-Swapped Antibodies:

Comparison of various activities was carried out in the followingmanner, on the purified samples of the various anti-CD20 domain-swappedantibodies obtained in the item 3 of Example 3.

1. CDC Activity of 1113-Type and 1131-Type Anti-CD20 Domain-SwappedAntibodies

In order to evaluate in vitro CDC activity of the CD20-IgG1-typeanti-CD20 human IgG1 chimeric antibody, CD20-IgG3-type anti-CD20 humanIgG3 chimeric antibody and 1133-type anti-CD20 domain-swapped antibodyobtained in the item 5 of Example 1 and the 1113-type anti-CD20domain-swapped antibody and 1131-type anti-CD20 domain-swapped antibodyobtained in the item 3 of Example 3, in a CD20-positive cell line, thetest was carried out in the same manner as in the item 2 of Example 2using a CD20-positive ST 486 cell or Raji cell.

The results are shown in FIG. 18. As shown in FIG. 18, the CDC activityof CD20-IgG3(−F) was higher than the CDC activity of CD20-IgG1(−F) ineach of the ST 486 cell line (FIG. 18A) and the Raji cell line (FIG.18B), and the CDC activity of 1133(−F) was higher than the CDC activityof CD20-IgG3(−F). In addition to this, the CDC activity of 1113(−F) and1131(−F) was higher than the CDC activity of CD20-IgG3(−F). Also, theCDC activity of 1131(−F) was higher than the CDC activity of 1113(−F).From these results, it was found that both of the CH2 domain and CH3domain from IgG3 are contributing to the increase of CDC activityeffected by the replacement of the Fc of IgG1 with the Fc of IgG3. Inaddition, it was found also from the above-described results thatcontribution of the CH2 domain is larger between the CH2 domain and CH3domain.

2. Evaluation of ADCC Activity for CD20-Positive Cell Line

In vitro ADCC activity of the anti-CD20 human IgG1 chimeric antibodyCD20-IgG1, anti-CD20 human IgG3 chimeric antibody CD20-IgG3 and1133-type anti-CD20 domain-swapped antibody obtained in the item 5 ofExample 1 and the 1113-type anti-CD20 domain-swapped antibody and1131-type anti-CD20 domain-swapped antibody obtained in the item 3 ofExample 3 was measured using a CD-positive Daudi cell as the target cellin accordance with the same procedure of the item 5 of Example 2.Cytotox 96™ Kit (manufactured by Promega) was used in the measurement.

The results are shown in FIG. 19. As shown in FIG. 19, 1113(−F) and1131(−F) also show ADCC activity equivalent to CD20-IgG1(−F) and1133(−F), and these results show that the ADCC activity is substantiallyequal to that of IgG1, even when the CH2 domain and/or CH3 domain of theanti-CD20 human IgG1 chimeric antibody is subjected to the domain-swapfor human IgG3.

Based on the above, it was confirmed that the 1113-type anti-CD20domain-swapped antibody and 1131-type anti-CD20 domain-swapped antibodyhaving the same variable region of the anti-CD20 human IgG1 chimericantibody, wherein only the CH2 domain or CH3 domain of the heavy chainconstant region contains the amino acid sequence from a human IgG3antibody and other domains contain the amino acid sequences from a humanIgG1 antibody, have CDC activity exceeding that of the anti-CD20 humanIgG3 chimeric antibody and ADCC activity equivalent to that of theanti-CD20 human IgG1 chimeric antibody.

Based on the results obtained in the above, relationship betweenstructures and activities of each of the prepared antibody anddomain-swapped antibodies is summarized in Table 6. In the table, ADCCactivity and CDC activity were expressed as ++++, +++, ++ and + in orderof the height of activities. In addition, regarding the binding activityto protein A, those having binding activity to protein A was shown by +,and having no activity as −.

TABLE 6 Structure ADCC CDC Protein A name CH1 Hinge CH2 CH3 activityactivity binding IgG1(−F) IgG1 IgG1 IgG1 IgG1 +++ + + IgG3(−F) IgG3 IgG3IgG3 IgG3 ++ ++ − 1133(+F)/ IgG1 IgG1 IgG3 IgG3 ++/+++ +++++ − 1133(−F)1113(−F) IgG1 IgG1 IgG1 IgG3 +++ +++ − 1131(−F) IgG1 IgG1 IgG3 IgG1 +++++++ +

Based on the above, it was found that the greater part of the high CDCactivity of an antibody molecule (1133-type domain-swapped antibody)having a heavy chain constant region in which the CH2 domain and the CH3domain in the human IgG1 antibody heavy chain constant region wereswapped for the amino acid sequence from a human IgG3 antibody is alsomaintained in an antibody molecule (1131-type domain-swapped antibody)having a heavy chain constant region in which only the CH2 domain in thehuman IgG1 antibody heavy chain constant region was swapped for theamino acid sequence from a human IgG3 antibody. In addition, it wasshown that the antibody molecule (1131-type domain-swapped antibody)having a heavy chain constant region in which only the CH2 domain in thehuman IgG1 antibody heavy chain constant region was replaced by theamino acid sequence from a human IgG3 antibody maintains high ADCCactivity equivalent to the human IgG1 antibody, and that the ADCCactivity is further enhanced when the fucose bound to theN-acetylglucosamine existing in the reducing terminal in the complextype N-glycoside-linked sugar chain bound to the Fc is removed.

Example 5 Measurement of Binding Activity of Anti-CD20 Domain-SwappedAntibodies to Various Recombinant Fcγ Receptors:

Binding activity of the anti-CD20 human IgG1 chimeric antibodiesCD20-IgG1(−F) and CD20-IgG1(+F) and 1133-type anti-CD20 domain-swappedantibodies 1133(−F) and 1133(+F) to Fc receptor family FcγRI and FcγRIIawas measured in accordance with a conventionally known method [Clin.Cancer Res., 10, 6248 (2004)].

The results are shown in FIG. 20. As shown in FIG. 20, the 1133-typeanti-CD20 domain-swapped antibodies showed their binding activity toFcγRI and also to FcγRIIa at similar level to that of the IgG1 anti-CD20antibodies. This result shows that replacement of CH2 and CH3 of theIgG1 antibodies by the amino acid sequence of the IgG3 antibodies doesnot influence on their binding activity to the Fc receptor family FcγRIand FcγRIIa.

In addition, as shown in FIG. 20, regardless of the presence or absenceof the fucose bound to the N-acetylglucosamine existing in the reducingterminal in the complex type N-glycoside-linked sugar chain bound to theFc, each antibody of the 1133-type anti-CD20 domain-swapped antibodiesand IgG1 anti-CD20 antibodies showed similar binding activity. The aboveresults show that the presence or absence of the fucose bound to theN-acetylglucosamine existing in the reducing terminal in the complextype N-glycoside-linked sugar chain bound to the Fc does not influenceon the binding activity to the Fc receptor family FcγRI and FcγRIIa, andthe activity is equivalent to that of the IgG1.

Example 6 Production of Various Anti-CD20 Domain-Swapped Antibodies inWhich a Polypeptide Containing the Human IgG1 Antibody CH2 Domain isReplaced by a Polypeptide Which Corresponds to the Human IgG3 AntibodyIndicated by the EU Index, Using Animal Cell:

1. Construction of Expression Vectors of Various Anti-CD20Domain-Swapped Antibodies in Which the Entire CH2 Domain and a Part ofCH3 Domain were Replaced by Amino Acid Sequences from Human IgG3Antibody

As seen in the item 1 of Example 4, it was found that replacement ofboth of the CH2 domain and the CH3 domain by the amino acid sequencesfrom an IgG3 antibody greatly contributes to the enhancement of CDCactivity of the human IgG1 antibody.

On the other hand, as seen in the item 5 of Example 1, the 1133-typeanti-CD20 domain-swapped antibody and 1113-type anti-CD20 domain-swappedantibody do not bind to protein A similarly to the case of the humanIgG3 antibody, but the 1131-type anti-CD20 domain-swapped antibody bindsto protein A similarly to the case of the human IgG1 antibody, and thisfact suggests that the CH3 domain containing the amino acid sequencefrom a human IgG1 antibody contributes to the binding to protein A.

When an antibody is produced as a medicament, it is important that theantibody has binding activity to protein A in view of purifying theantibody easily. Accordingly, domain-swapped antibodies which have CDCactivity equivalent to the 1133-type and also have binding activity toprotein A were purified by completely replacing the CH2 domain from ahuman IgG1 with the CH2 domain from a human IgG3 antibody and partiallyreplacing the CH3 domain of IgG1 with the CH3 domain from a human IgG3antibody.

A schematic illustration of heavy chain constant regions of variousanti-CD20 domain-swapped antibodies designed in this Example is shown inFIG. 21. Since amino acid sequences of the heavy chain constant regionsof these domain-swapped antibodies are unknown, each of them is a novelstructure. The CH2 domain of the IgG antibody contains the amino acidresidues at positions 231 to 340 indicated by the EU index, and the CH3domain thereof contains the amino acid residues at positions 341 to 447indicated by the EU index.

The 113A-type anti-CD20 domain-swapped antibody is a domain-swappedantibody in which a polypeptide comprising the CH2 domain in the heavychain constant region of the anti-CD20 human IgG1 chimeric antibody isreplaced by a polypeptide corresponding to positions 231 to 356 of ahuman IgG3 antibody indicated by the EU index.

The 113B-type anti-CD20 domain-swapped antibody is a domain-swappedantibody in which a polypeptide comprising the CH2 domain in the heavychain constant region of the anti-CD20 human IgG1 chimeric antibody isreplaced by a polypeptide corresponding to positions 231 to 358 of ahuman IgG3 antibody indicated by the EU index.

The 113C-type anti-CD20 domain-swapped antibody is a domain-swappedantibody in which a polypeptide comprising the CH2 domain in the heavychain constant region of the anti-CD20 human IgG1 chimeric antibody isreplaced by a polypeptide corresponding to positions 231 to 384 of ahuman IgG3 antibody indicated by the EU index.

The 113D-type anti-CD20 domain-swapped antibody is a domain-swappedantibody in which a polypeptide comprising the CH2 domain in the heavychain constant region of the anti-CD20 human IgG1 chimeric antibody isreplaced by a polypeptide corresponding to positions 231 to 392 of ahuman IgG3 antibody indicated by the EU index.

The 113E-type anti-CD20 domain-swapped antibody is a domain-swappedantibody in which a polypeptide comprising the CH2 domain in the heavychain constant region of the anti-CD20 human IgG1 chimeric antibody isreplaced by a polypeptide corresponding to positions 231 to 397 of ahuman IgG3 antibody indicated by the EU index.

The 113F-type anti-CD20 domain-swapped antibody is a domain-swappedantibody in which a polypeptide comprising the CH2 domain in the heavychain constant region of the anti-CD20 human IgG1 chimeric antibody isreplaced by a polypeptide corresponding to positions 231 to 422 of ahuman IgG3 antibody indicated by the EU index.

The 113G-type anti-CD20 domain-swapped antibody is a domain-swappedantibody in which a polypeptide comprising the CH2 domain in the heavychain constant region of the anti-CD20 human IgG1 chimeric antibody isreplaced by polypeptides corresponding to positions 231 to 434 andpositions 436 to 447 of a human IgG3 antibody indicated by the EU index.

The 113H-type anti-CD20 domain-swapped antibody is a domain-swappedantibody in which a polypeptide comprising the CH2 domain in the heavychain constant region of the anti-CD20 human IgG1 chimeric antibody isreplaced by a polypeptide corresponding to positions 231 to 435 of ahuman IgG3 antibody indicated by the EU index.

These various anti-CD20 domain-swapped antibodies were prepared by thefollowing procedure.

Each of these anti-CD20 domain-swapped antibodies can be produced bypreparing a DNA fragment encoding the amino acid sequence of the CH3domain of each domain-swapped antibody, and replacing it with anucleotide sequence of the expression vector for 1133-type anti-CD20domain-swapped antibody, pKTX93/1133 prepared in the item 2 of Example2, encoding the amino acid sequence of the CH3 domain thereof.Replacement of the nucleotide sequence encoding the heavy chain CH3domain can be carried out using a restriction enzyme recognitionsequence Bsp1407I positioned at the 5′-terminal side in the nucleotidesequence encoding the heavy chain CH3 domain and a restriction enzymerecognition sequence NruI positioned at the 3′-terminal side in thenucleotide sequence encoding the heavy chain CH3 domain.

(1) Construction of Expression Vector Comprising the Nucleotide SequenceEncoding 113A-Type Anti-CD20 Domain-Swapped Antibody

An expression vector comprising the nucleotide sequence of the 113A-typedomain-swapped antibody in which a polypeptide comprising the CH2 domainin the heavy chain constant region of anti-CD20 human IgG1 chimericantibody was replaced by a polypeptide corresponding to positions 231 to356 of a human IgG3 antibody indicated by the EU index was constructedby the procedure shown below (FIG. 22). The amino acid sequence of theheavy chain constant region of 113A-type anti-CD20 domain-swappedantibody is shown in SEQ ID NO:33.

Firstly, the nucleotide sequence represented by SEQ ID NO:34 wasdesigned. The sequence was designed based on the sequence of arestriction enzyme recognition sequence Bsp1407I positioned at the5′-terminal side in the nucleotide sequence encoding the heavy chain CH3domain to a restriction enzyme recognition sequence NruI positioned atthe 3′-terminal side in the nucleotide sequence encoding the heavy chainCH3 domain, on the expression vector for 1133-type anti-CD20domain-swapped antibody prepared in the item 2 of Example 2, and amongthe amino acid sequences encoded by the nucleotide sequences, the aminoacid sequence of the N-terminal side to position 356 indicated by the EUindex was based on the amino acid sequence from a human IgG3 antibody,and the amino acid sequence at positions 357 to 447 indicated by the EUindex was based on the amino acid sequence from a human IgG1 antibody.Next, each of the nucleotide sequences represented by SEQ ID NOs:35 and36 was designed. The nucleotide sequences represented by SEQ ID NOs:35and 36 are the nucleotide sequences of a sense primer and an antisenseprimer, respectively, for amplifying a DNA fragment consisting of thenucleotide sequence represented by SEQ ID NO:34 by PCR. Each ofsynthetic oligo DNAs of the nucleotide sequences represented by SEQ IDNOs:35 and 36 was prepared (manufactured by FASMAC), and PCR was carriedout using, as the template, the expression vector plasmid of 1133-typeanti-CD20 domain-swapped antibody prepared in the item 2 of Example 2.By preparing a reaction solution for PCR [0.05 unit/μl KOD DNAPolymerase (manufactured by TOYOBO), 0.2 mM dNTPs, 1 mM magnesiumchloride, 1/10 volume of 10-fold concentrated PCR Buffer #2(manufactured by TOYOBO, attached to the KOD DNA Polymerase)] in such amanner that each of the two synthetic oligo DNA become the finalconcentration of 0.5 μM, and PCR was carried out using a DNA thermalcycler GeneAmp PCR System 9700™ (manufactured by Applied Biosystems) byheating at 94° C. for 4 minutes, followed by 25 cycles consisting of 3steps of reactions at 94° C. for 30 seconds, at 55° C. for 30 secondsand at 74° C. for 60 seconds. After completion of the PCR, the reactionsolution was subjected to agarose gel electrophoresis, and a PCR productof about 300 bp was recovered using QIAquick™ Gel Extraction Kit(manufactured by QIAGEN). The thus recovered PCR product was digestedwith a restriction enzyme Bsp1407I (manufactured by Takara Shuzo) and arestriction enzyme NruI (manufactured by Takara Shuzo), and then thereaction solution was subjected to agarose gel electrophoresis, and aDNA fragment of about 300 bp was cleaved and purified using QIAquick™Gel Extraction Kit (manufactured by QIAGEN). On the other hand, a DNAfragment of about 13 kbp was cleaved and purified by the same treatmentwith restriction enzymes on the expression vector plasmid of 1133-typeanti-CD20 domain-swapped antibody prepared in the item 2 of Example 2.After mixing these purified DNA fragments, a ligation reaction wascarried out by adding Ligation High solution (manufactured by TOYOBO),and Escherichia coli XL1-BLUE MRF′ (manufactured by Stratagene) wastransformed using the reaction solution. Each plasmid DNA was preparedfrom the thus obtained transformant clones and allowed to react usingBig Dye Terminator Cycle™ Sequencing Kit v3.1 (manufactured by AppliedBiosystems) in accordance with the instructions attached thereto, andthen the nucleotide sequence of the DNA inserted into each plasmid wasanalyzed by a DNA sequencer ABI PRISM 3700™ DNA Analyzer of the samecompany to confirm that expression vector plasmid for 113A-typeanti-CD20 domain-swapped antibody, pKTX93/113A was obtained.

(2) Construction of Expression Vector Comprising the Nucleotide SequenceEncoding 113B-Type Anti-CD20 Domain-Swapped Antibody

An expression vector comprising the nucleotide sequence of the 113B-typedomain-swapped antibody in which a polypeptide containing the CH2 domainin the heavy chain constant region of anti-CD20 human IgG1 chimericantibody was replaced by a polypeptide corresponding to positions 231 to358 of a human IgG3 antibody indicated by the EU index was constructedby the procedure shown below (FIG. 22). The amino acid sequence of theheavy chain constant region of 113B-type anti-CD20 domain-swappedantibody is shown in SEQ ID NO:37.

Firstly, the nucleotide sequence represented by SEQ ID NO:38 wasdesigned. The sequence was designed based on the sequence of arestriction enzyme recognition sequence Bsp1407I positioned at the5′-terminal side in the nucleotide sequence encoding the heavy chain CH3domain to a restriction enzyme recognition sequence NruI positioned atthe 3′-terminal side in the nucleotide sequence encoding the heavy chainCH3 domain, on the expression vector for 1133-type anti-CD20domain-swapped antibody prepared in the item 2 of Example 2, and amongthe amino acid sequences encoded by the nucleotide sequences, the aminoacid sequence of the N-terminal side to position 358 indicated by the EUindex was based on the amino acid sequence from a human IgG3 antibody,and the amino acid sequence at positions 359 to 447 indicated by the EUindex was based on the amino acid sequence from a human IgG1 antibody.Next, the nucleotide sequence represented by SEQ ID NO:39 was designed.The nucleotide sequence represented by SEQ ID NO:39 is the nucleotidesequence of a sense primer for use in the amplification of a DNAfragment containing the nucleotide sequence represented by SEQ ID NO:38by PCR, and was used in combination with an antisense primer containingthe nucleotide sequence represented by SEQ ID NO:36. Each of syntheticoligo DNAs of the nucleotide sequences represented by SEQ ID NOs:39 and36 was prepared (manufactured by FASMAC), and PCR was carried out using,as the template, the expression vector plasmid for 1133-type anti-CD20domain-swapped antibody prepared in the item 2 of Example 2. Thereafter,expression vector plasmid for 113B-type anti-CD20 domain-swappedantibody, pKTX93/113B was prepared in the same manner as in the (1) ofthis item.

(3) Construction of Expression Vector Comprising the Nucleotide SequenceEncoding 113C-Type Anti-CD20 Domain-Swapped Antibody

An expression vector comprising the nucleotide sequence of the 113C-typedomain-swapped antibody in which a polypeptide comprising the CH2 domainin the heavy chain constant region of anti-CD20 human IgG1 chimericantibody was replaced by a polypeptide corresponding to positions 231 to384 of a human IgG3 antibody indicated by the EU index was constructedby the procedure shown below (FIG. 22). The amino acid sequence of theheavy chain constant region of 113C-type anti-CD20 domain-swappedantibody is shown in SEQ ID NO:40.

Firstly, the nucleotide sequence represented by SEQ ID NO:41 wasdesigned. The sequence was designed based on the sequence of arestriction enzyme recognition sequence Bsp1407I positioned at the5′-terminal side in the nucleotide sequence encoding the heavy chain CH3domain to a restriction enzyme recognition sequence NruI positioned atthe 3′-terminal side in the nucleotide sequence encoding the heavy chainCH3 domain, on the expression vector for 1133-type anti-CD20domain-swapped antibody prepared in the item 2 of Example 2, and amongthe amino acid sequences encoded by the nucleotide sequences, the aminoacid sequence of the N-terminal side to position 384 indicated by the EUindex was based on the amino acid sequence from a human IgG3 antibody,and the amino acid sequence at positions 385 to 447 indicated by the EUindex was based on the amino acid sequence from a human IgG1 antibody.Next, each of the nucleotide sequences represented by SEQ ID NOs:42 and43 was designed. The nucleotide sequences represented by SEQ ID NOs:42and 43 are nucleotide sequences of synthetic oligo DNA for amplifying aDNA fragment containing the nucleotide sequence represented by SEQ IDNO:41 by PCR. The 3′-terminal side of the nucleotide sequencerepresented by SEQ ID NO:42 and the 5′-terminal side of the nucleotidesequence represented by SEQ ID NO:43 were designed in such a manner thatapproximately 20 bps thereof were mutually overlapped like acomplementary sequence so that annealing was caused when PCR was carriedout. Each of synthetic oligo DNAs of the nucleotide sequencesrepresented by SEQ ID NOs:42 and 43 was prepared (manufactured byFASMAC), and PCR was carried out. By preparing a reaction solution forPCR [0.02 unit/μl KOD+DNA Polymerase (manufactured by TOYOBO), 0.2 mMdNTPs, 1 mM magnesium chloride, 1/10 volume of 10-fold concentrated PCRBuffer (manufactured by TOYOBO, attached to the KOD+DNA Polymerase)] insuch a manner that each of the two synthetic oligo DNAs become the finalconcentration of 0.2 μM, and PCR was carried out using a DNA thermalcycler GeneAmp PCR System 9700 (manufactured by Applied Biosystems) byheating at 94° C. for 4 minutes, followed by 25 cycles of 3 steps ofreactions at 94° C. for 30 seconds, at 55° C. for 30 seconds and 68° C.for 60 seconds. Thereafter, expression vector plasmid for 113C-typeanti-CD20 domain-swapped antibody, pKTX93/113C was prepared in the samemanner as in the (1) of this item.

(4) Construction of Expression Vector Comprising the Nucleotide SequenceEncoding 113D-Type Anti-CD20 Domain-Swapped Antibody

An expression vector comprising the nucleotide sequence of 113D-typedomain-swapped antibody in which a polypeptide comprising the CH2 domainin the heavy chain constant region of anti-CD20 human IgG1 chimericantibody was replaced by a polypeptide corresponding to positions 231 to392 of a human IgG3 antibody indicated by the EU index was constructedby the procedure shown below (FIG. 22). The amino acid sequence of theheavy chain constant region of 113D-type anti-CD20 domain-swappedantibody is shown in SEQ ID NO:44.

Firstly, the nucleotide sequence represented by SEQ ID NO:45 wasdesigned. The sequence was designed based on the sequence of arestriction enzyme recognition sequence Bsp1407I positioned at the5′-terminal side in the nucleotide sequence encoding the heavy chain CH3domain to a restriction enzyme recognition sequence NruI positioned atthe 3′-terminal side in the nucleotide sequence encoding the heavy chainCH3 domain, on the expression vector for 1133-type anti-CD20domain-swapped antibody prepared in the item 2 of Example 2, and amongthe amino acid sequences encoded by the nucleotide sequences, the aminoacid sequence of the N-terminal side to position 392 indicated by the EUindex was based on the amino acid sequence from a human IgG3 antibody,and the amino acid sequence at positions 393 to 447 indicated by the EUindex was based on the amino acid sequence from a human IgG1 antibody.Next, the nucleotide sequence represented by SEQ ID NO:46 was designed.The nucleotide sequence represented by SEQ ID NO:46 is the nucleotidesequence of a synthetic oligo DNA for use in the amplification of a DNAfragment containing the nucleotide sequence represented by SEQ ID NO:45by PCR, which is used in combination with a synthetic oligo DNAcontaining the nucleotide sequence represented by SEQ ID NO:43. The3′-terminal side of the nucleotide sequence represented by SEQ ID NO:46and the 5′-terminal side of the nucleotide sequence represented by SEQID NO:43 were designed in such a manner that approximately 20 bpsthereof were mutually overlapped so that annealing was caused when PCRwas carried out. Each of synthetic oligo DNAs of the nucleotidesequences represented by SEQ ID NOs:46 and 43 was prepared (manufacturedby FASMAC), and PCR was carried out. Thereafter, expression vectorplasmid for 113D-type anti-CD20 domain-swapped antibody, pKTX93/113D wasprepared in the same manner as in (3) of this item.

(5) Construction of Expression Vector Comprising the Nucleotide SequenceEncoding 113E-Type Anti-CD20 Domain-Swapped Antibody

An expression vector comprising the nucleotide sequence of thedomain-swapped antibody 113E-type in which a polypeptide comprising theCH2 domain in the heavy chain constant region of anti-CD20 human IgG1chimeric antibody was replaced by a polypeptide corresponding topositions 231 to 397 of a human IgG3 antibody indicated by the EU indexwas constructed by the procedure shown below (FIG. 22). The amino acidsequence of the heavy chain constant region of 113E-type anti-CD20domain-swapped antibody is shown in SEQ ID NO:47.

Firstly, the nucleotide sequence represented by SEQ ID NO:48 wasdesigned. The sequence was designed based on the sequence of arestriction enzyme recognition sequence Bsp1407I positioned at the5′-terminal side in the nucleotide sequence encoding the heavy chain CH3domain to a restriction enzyme recognition sequence NruI positioned atthe 3′-terminal side in the nucleotide sequence encoding the heavy chainCH3 domain, on the expression vector for 1133-type anti-CD20domain-swapped antibody prepared in the item 2 of Example 2, and amongthe amino acid sequences encoded by the nucleotide sequences, the aminoacid sequence of the N-terminal side to position 397 indicated by the EUindex was based on the amino acid sequence from a human IgG3 antibody,and the amino acid sequence at positions 398 to 447 indicated by the EUindex was based on the amino acid sequence from a human IgG1 antibody.Next, the nucleotide sequences represented by SEQ ID NO:49 was designed.The nucleotide sequence represented by SEQ ID NO:49 is the nucleotidesequence of a synthetic oligo DNA for use in the amplification of a DNAfragment containing the nucleotide sequence represented by SEQ ID NO:48by PCR, which is used in combination with a synthetic oligo DNAcontaining the nucleotide sequence represented by SEQ ID NO:43. The3′-terminal side of the nucleotide sequence represented by SEQ ID NO:49and the 5′-terminal side of the nucleotide sequence represented by SEQID NO:43 were designed in such a manner that approximately 20 bpsthereof were mutually overlapped so that annealing was caused when PCRwas carried out. Each of synthetic oligo DNAs of the nucleotidesequences represented by SEQ ID NOs:49 and 43 was prepared (manufacturedby FASMAC), and PCR was carried out. Thereafter, expression vectorplasmid for 113E-type anti-CD20 domain-swapped antibody, pKTX93/113E wasprepared in the same manner as in (3) of this item.

(6) Construction of Expression Vector Comprising the Nucleotide SequenceEncoding 113F-Type Anti-CD20 Domain-Swapped Antibody

An expression vector comprising the nucleotide sequence of the 113F-typedomain-swapped antibody in which a polypeptide comprising the CH2 domainin the heavy chain constant region of anti-CD20 human IgG1 chimericantibody was replaced by a polypeptide corresponding to positions 231 to422 of a human IgG3 antibody indicated by the EU index was constructedby the procedure shown below (FIG. 22). The amino acid sequence of theheavy chain constant region of 113F-type anti-CD20 domain-swappedantibody is shown in SEQ ID NO:50.

Firstly, the nucleotide sequence represented by SEQ ID NO:51 wasdesigned. The sequence was designed based on the sequence of arestriction enzyme recognition sequence Bsp1407I positioned at the5′-terminal side in the nucleotide sequence encoding the heavy chain CH3domain to a restriction enzyme recognition sequence NruI positioned atthe 3′-terminal side in the nucleotide sequence encoding the heavy chainCH3 domain, on the expression vector for 1133-type anti-CD20domain-swapped antibody prepared in the item 2 of Example 2, and amongthe amino acid sequences encoded by the nucleotide sequences, the aminoacid sequence of the N-terminal side to position 422 indicated by the EUindex was based on the amino acid sequence from a human IgG3 antibody,and the amino acid sequence at positions 423 to 447 indicated by the EUindex was based on the amino acid sequence from a human IgG1 antibody.Each of synthetic oligo DNAs of the nucleotide sequences represented bySEQ ID NOs:39 and 36 was prepared (manufactured by FASMAC), and PCR wascarried out using, as the template, the expression vector plasmid foranti-CD20 human IgG3 chimeric antibody, pKANTEX2B8γ3 prepared in theitem 1 of Example 1. Thereafter, expression vector plasmid for 113F-typeanti-CD20 domain-swapped antibody, pKTX93/113F was prepared in the samemanner as in (1) of this item.

(7) Construction of Expression Vector Comprising the Nucleotide SequenceEncoding 113H-Type Anti-CD20 Domain-Swapped Antibody

An expression vector comprising the nucleotide sequence of the 113H-typedomain-swapped antibody in which a polypeptide comprising the CH2 domainin the heavy chain constant region of anti-CD20 human IgG1 chimericantibody was replaced by a polypeptide corresponding to positions 231 to435 of a human IgG3 antibody indicated by the EU index was constructedby the procedure shown below (FIG. 22). The amino acid sequence of theheavy chain constant region of 113H-type anti-CD20 domain-swappedantibody is shown in SEQ ID NO:52.

Firstly, the nucleotide sequence represented by SEQ ID NO:53 wasdesigned. The sequence was designed based on the sequence of arestriction enzyme recognition sequence Bsp1407I positioned at the5′-terminal side in the nucleotide sequence encoding the heavy chain CH3domain to a restriction enzyme recognition sequence NruI positioned atthe 3′-terminal side in the nucleotide sequence encoding the heavy chainCH3 domain, on the expression vector for 1133-type anti-CD20domain-swapped antibody prepared in the item 2 of Example 2, and amongthe amino acid sequences encoded by the nucleotide sequences, the aminoacid sequence of the N-terminal side to position 435 indicated by the EUindex was based on the amino acid sequence from a human IgG3 antibody,and the amino acid sequence at positions 436 to 447 indicated by the EUindex was based on the amino acid sequence from a human IgG1 antibody.Next, the nucleotide sequence represented by SEQ ID NO:54 was designed.The nucleotide sequence represented by SEQ ID NO:54 is a nucleotidesequence of the antisense primer to be used in the amplification of aDNA fragment containing the nucleotide sequence represented by SEQ IDNO:53 by PCR, which is used in combination with the sense primercontaining the nucleotide sequence represented by SEQ ID NO:39. Each ofsynthetic oligo DNAs of the nucleotide sequences represented by SEQ IDNOs:39 and 54 was prepared (manufactured by FASMAC), and PCR was carriedout using, as the template, the expression vector plasmid for anti-CD20human IgG3 chimeric antibody, pKANTEX2B8γ3 prepared in the item 1 ofExample 1. Thereafter, expression vector plasmid for 113H-type anti-CD20domain-swapped antibody, pKTX93/113H was prepared in the same manner asin (1) of this item.

(8) Construction of Expression Vector Comprising the Nucleotide SequenceEncoding 113G-Type Anti-CD20 Domain-Swapped Antibody

An expression vector comprising the nucleotide sequence of the 113G-typedomain-swapped antibody in which a polypeptide containing the CH2 domainin the heavy chain constant region of anti-CD20 human IgG1 chimericantibody was replaced by a polypeptide corresponding to positions 231 to434 of a human IgG3 antibody indicated by the EU index was constructedby the procedure shown below (FIG. 22). The amino acid sequence of theheavy chain constant region of 113G-type anti-CD20 domain-swappedantibody is shown in SEQ ID NO:55.

Firstly, the nucleotide sequence represented by SEQ ID NO:56 wasdesigned. The sequence was designed based on the sequence of arestriction enzyme recognition sequence Bsp1407I positioned at the5′-terminal side in the nucleotide sequence encoding the heavy chain CH3domain to a restriction enzyme recognition sequence NruI positioned atthe 3′-terminal side in the nucleotide sequence encoding the heavy chainCH3 domain, on the expression vector for 1133-type anti-CD20domain-swapped antibody prepared in the item 2 of Example 2, and amongthe amino acid sequences encoded by the nucleotide sequences, the aminoacid sequence of the N-terminal side to position 434 indicated by the EUindex was based on the amino acid sequence from a human IgG3 antibody,the amino acid sequence at position 435 indicated by the EU index wasbased on the amino acid sequence from a human IgG1 antibody, and theamino acid sequence at positions 436 to 447 indicated by the EU indexwas based on the amino acid sequence from a human IgG3 antibody. Next,the nucleotide sequence represented by SEQ ID NO:57 was designed. Thenucleotide sequence represented by SEQ ID NO:57 is a nucleotide sequenceof the antisense primer to be used in the amplification of a DNAfragment containing the nucleotide sequence represented by SEQ ID NO:56by PCR, which is used in combination with the sense primer containingthe nucleotide sequence represented by SEQ ID NO:39. Each of syntheticoligo DNAs of the nucleotide sequences represented by SEQ ID NOs:39 and56 was prepared (manufactured by FASMAC), and PCR was carried out using,as the template, the expression vector plasmid for anti-CD20 human IgG3chimeric antibody, pKANTEX2B8γ3 prepared in the item 1 of Example 1.Thereafter, expression vector plasmid for 113G-type anti-CD20domain-swapped antibody, pKTX93/113G was prepared in the same manner asin (1) of this item.

2. Stable Expression of Various Anti-CD20 Domain-Swapped Antibodies inWhich the Entire CH2 Domain and a Part of CH3 Domain were Replaced bythe Amino Acid Sequences from a Human IgG3 Antibody, in Animal Cell

A cell capable of stably producing each of various anti-CD20domain-swapped antibodies in which the entire CH2 domain and a part ofCH3 domain were replaced by the amino acid sequences from a human IgG3antibody was prepared in the same manner as in the item 3 of Example 1,by introducing each of the expression vectors of anti-CD20domain-swapped antibodies in which the entire CH2 domain and a part ofCH3 domain were replaced by the amino acid sequences from a human IgG3antibody, prepared in the item 1 of this Example, into the host cellCHO/FUT8^(−/−) described in the item 3 of Example 1.

3. Purification of Various Anti-CD20 Domain-Swapped Antibodies in Whichthe entire CH2 Domain and a Part of the CH3 Domain were Replaced by theAmino Acid Sequence from a Human IgG3 Antibody:

Each of the transformants obtained in the item 2 of this Example capableof expressing various anti-CD20 domain-swapped antibodies in which theentire CH2 domain and a part of the CH3 domain were replaced by theamino acid sequences from a human IgG3 antibody was cultured andpurified in the same manner as in the item 5 of Example 1. Prosep-Gcolumn was used in the purification. Corresponding expression vector,host cell, name of the purified antibody and amino acid sequence ofheavy chain constant region of each of the modified antibodies are shownin Table 7.

TABLE 7 Purified antibody Amino acid Expression vector Host cell (name)sequence PKTX93/113A Ms705 113A(−F) SEQ ID NO: 33 PKTX93/113B Ms705113B(−F) SEQ ID NO: 37 PKTX93/113C Ms705 113C(−F) SEQ ID NO: 40PKTX93/113D Ms705 113D(−F) SEQ ID NO: 44 PKTX93/113E Ms705 113E(−F) SEQID NO: 47 PKTX93/113F Ms705 113F(−F) SEQ ID NO: 50 PKTX93/113G Ms705113G(−F) SEQ ID NO: 55 PKTX93/113H Ms705 113H(−F) SEQ ID NO: 52

4. Evaluation of the Purification Degree of Various Anti-CD20Domain-Swapped Antibodies by SDS-PAGE

In order to evaluate purification degree of the purified samples ofvarious anti-CD20 domain-swapped antibodies obtained in the item 3 ofthis Example, SDS-PAGE was carried out in the same manner as in the item6 of Example 1. As comparative controls of the electrophoresis, the sameoperation was carried out also on the purified samples CD20-IgG1(−F) and1133(−F) prepared in the item 5 of Example 1.

The results are shown in FIG. 23. Each of purified samples of theanti-CD20 domain-swapped antibodies obtained in the item 3 of thisExample showed similar electrophoresis patterns of the CD20-IgG1(−F) and1133(−F). The molecular weights deduced from the amino acid sequences ofH chain and L chain constituting each of the various anti-CD20domain-swapped antibodies was similar to each other, namely the H chainwas about 50 kilodaltons (hereinafter referred to as kDa) and the Lchain was about 24 kDa. Since these molecular weights are similar to themolecular weights of the H chain and L chain of CD20-IgG1(−F) and1133(−F) and their electrophoresis patterns are also similar thereto, itwas confirmed that each of the various anti-CD20 domain-swapped antibodyis constituted by the desired H chain and L chain.

Based on the above results, it was confirmed that the desired IgGmolecules respectively constituted by the H chain and L chain arecontained at a sufficient ratio in the purified samples of variousanti-CD20 domain-swapped antibodies obtained in the item 3 of thisExample.

Example 7

Activity Evaluation of Various Anti-CD20 Domain-Swapped Antibodies inWhich the Entire CH2 Domain and a Part of CH3 Domain were Replaced byAmino Acid Sequences from Human IgG3 Antibody:

Comparison of various activities was carried out for the purifiedsamples of various anti-CD20 domain-swapped antibodies obtained in theitem 3 of Example 6 in the following manner.

1. Measurement of the CDC Activity of Various Anti-CD20 Domain-SwappedAntibodies in Which the Entire CH2 Domain and a Part of CH3 Domain wereReplaced by Amino Acid Sequences from Human IgG3 Antibody

The in vitro CDC activity in a human CD20 gene-introduced cell lineCD20/EL4-A [Clin. Cancer Res., 11, 2327 (2005)] was measured for thepurified samples of various anti-CD20 domain-swapped antibodies obtainedin the item 3 of Example 6, the 1133-type anti-CD20 domain-swappedantibody obtained in the item 5 of Example 1 and the 1131-type anti-CD20domain-swapped antibody obtained in the item 3 of Example 3. Thereaction was carried out in a 96-well flat-bottomed plate (manufacturedby Sumitomo Bakelite), and a human complement dilution medium containing5×10⁴ cells of the target cell and containing each anti-CD20domain-swapped antibody at varied concentrations (0.1 μg/ml to 30 μg/ml)was dispensed at 150 μl into each reaction well. Thereafter, the testwas carried out in the same manner as in the item 2 of Example 2.

The results are shown in FIG. 24. All of the various anti-CD20domain-swapped antibodies showed CDC activity of similar to or higherthan that of the 1131(−F), particularly, the 113E(−F), 113F(−F),113G(−F) and 113H(−F) showed strongly higher CDC activity than that ofthe 1131(−F).

2. Measurement of the Binding Activity to Protein A of Various Anti-CD20Domain-Swapped Antibodies in Which the Entire CH2 Domain and a Part ofCH3 Domain were Replaced by Amino Acid Sequences from Human IgG3Antibody

The binding activity to protein A was measured by the proceduredescribed below of the purified samples of various anti-CD20domain-swapped antibodies obtained in the item 3 of Example 6, theCD20-IgG1(−F), CD20-IgG3(−F) and 113(−F) obtained in the item 5 ofExample 1 and the 1131(−F) and 1113(−F) obtained in the item 3 ofExample 3.

A goat anti-human kappa chain antibody (manufactured by Sigma-Aldrich)was diluted with PBS to a concentration of 5 μg/ml, dispensed at 50μl/well into a 96-well plate for ELISA (manufactured by Greiner) andthen allowed to stand at room temperature for 1 hour for adsorption.After the reaction and subsequent washing with PBS, 1% BSA-PBS was addedthereto at 100 μl/well and allowed to react at room temperature for 1hour for blocking the remaining active groups. After removing the 1%BSA-PBS, each antibody to be measured was added thereto at 50 μl/well atvaried concentrations (0.01 μg/ml to 10 μg/ml) and allowed to react atroom temperature for 2 hours. After the reaction and subsequent washingof each well with Tween-PBS, a peroxidase-labeled protein A solution(manufactured by Amersham Bioscience) diluted 5,000-fold with PBS wasadded at 50 μl/well and allowed to react at 37° C. for 2 hours. Afterwashing with Tween-PBS, the ABTS substrate solution was added at 50μl/well for color development, and then OD415 was measured.

The results are shown in FIG. 25. Firstly, the binding activity toprotein A was compared with CD20-IgG1(−F), CD20-IgG3(−F), 1133(−F),1131(−F) and 1113(−F) (FIG. 25A). As shown in FIG. 25A, both ofCD20-IgG1(−F) and 1131(−F) showed binding activity to protein Adepending on concentration, and the activity levels are equivalent toeach other. In the case of CD20-IgG3(−F), 1133(−F) and 1113(−F), on theother hand, the binding activity to protein A was not found within themeasured concentration range (10 μg/ml or less).

Next, the binding activity to protein A of various anti-CD20domain-swapped antibodies was compared with that of CD20-IgG1(−F) and1131(−F). As shown in FIG. 25B, 1133(−F) and 113H(−F) did not showbinding activity to protein A, but 113A(−F), 113B(−F), 113C(−F),113D(−F), 113E(−F), 113F(−F) and 113G(−F) showed binding activity toprotein A of equivalent to that of IgG1.

Grades of the CDC activity and protein A binding activity of variousanti-CD20 domain-swapped antibodies are shown in Table 8.

TABLE 8 Antibody CDC activity Binding activity to protein ACD20-IgG1(−F) + + 1131(−F) ++ + 113A(−F) ++ + 113B(−F) ++ + 113C(−F)++ + 113D(−F) ++ + 113E(−F) +++ + 113F(−F) ++++ + 113G(−F) +++ +113H(−F) ++++ − 1133(−F) ++++ −

In the 1133-type domain-swapped antibody in which CH2 and CH3 of IgG1antibody were replaced by the amino acid sequences of IgG3, its CDCactivity was enhanced, but the binding activity to protein A wasdeleted. On the other hand, in the 1131-type antibody in which CH2 ofIgG1 antibody alone was replaced by the amino acid sequence of IgG3, itmaintained the binding activity to protein A but the CDC activityenhancing ratio was reduced. Regarding the various anti-CD20domain-swapped antibodies prepared in this Example, in which the entireCH2 domain and a part of CH3 domain were replaced by the correspondingamino acid sequences from a human IgG3 antibody, all of them excluding113H(−F) have CDC activity and binding activity to protein A which werehigher than those of IgG1. In addition, 113E(−F), 113F(−F) and 113G(−F)having a relatively high ratio of the amino acid sequence from a humanIgG3 antibody occupying the whole CH3 domain showed higher CDC activitythan that of 1131(−F) and had binding activity to protein A similar tothat of the human IgG1 antibody. Among the anti-CD20 domain-swappedantibodies having similar binding activity to protein A to that of thehuman IgG1 antibody, 113F(−F) showed particularly high CDC activity.

Based on the above, it was found that, in the antibodies in which theentire CH2 domain of IgG1 antibody was replaced by the CH2 domain froman IgG3 antibody and a part of the CH3 domain was replaced by the CH3domain from an IgG3 antibody, the CDC activity was enhanced to a levelgreater than that of the antibodies in which the CH2 domain from an IgG1antibody alone was replaced by the CH2 domain derived from an IgG3antibody, and they can maintain the binding activity to protein Asimilar to that of the human IgG1 antibody.

Example 8 Evaluation of the CDC Activity of Various Anti-CD20Domain-Swapped Antibodies for Chronic Lymphocytic Leukemia (CLL) Cells

Using the CD20-IgG1(−F) obtained in the item 5 of Example 1, 1133(−F)obtained in the item 5 of Example 1, 1131(−F) obtained in the item 3 ofExample 3 and 113F(−F) obtained in the item 3 of Example 6, in vitro CDCactivity for CD20-positive CLL cell lines MEC-1 (DSMZ: ACC 497), MEC-2(DSMZ: ACC 500) and EHEB (human, peripheral blood, leukemia, chronic, Bcell) (DSMZ: ACC 67) was measured. The reaction was carried out in a96-well flat-bottomed plate (manufactured by Sumitomo Bakelite), and ahuman complement dilution medium containing 5×10⁴ cells of the targetcell and containing each anti-CD20 antibody at varied concentrations(from 0.04 μg/ml to 100 μg/ml) was dispensed at 150 μl into eachreaction well. Thereafter, the test was carried out in the same manneras in the item 2 of Example 2.

The results are shown in FIG. 26. In comparison with the CD20-IgG1, CDCactivity of 1133(−F), 1131(−F) and 113F(−F) was significantly enhancedfor all of the CD20-positive CLL cell lines MEC-1 (FIG. 26A), MEC-2(FIG. 26B) and EHEB (FIG. 26C). The above results suggest thatmedicaments containing each of these antibodies as an active ingredientare effective for the treatment of CLL.

Example 9 Preparation of Anti-Campath Human IgG1 Antibody, 1133-TypeAnti-Campath Domain-Swapped Antibody and 1131-Type Anti-CampathDomain-Swapped Antibody 1. Construction of Expression Vectors ofAnti-Campath Human IgG1 Antibody, 1133-Type Anti-Campath Domain-SwappedAntibody and 1131-Type Anti-Campath Domain-Swapped Antibody

In the comparison of the CDC activity of anti-CD20 domain-swappedantibodies 1131(−F) and 1113(−F) carried out in the item 1 of Example 4,both of the 1131(−F) and 1113(−F) showed higher CDC activity than thatof the IgG1, and particularly, the 1131(−F) showed higher CDC activitythan that of the 1113(−F), and it was found that when the CH2 domain wasIgG3, it greatly contributes to the enhancement of CDC activity. Inorder to confirm that similar CDC activity enhancement can also be foundin the antibodies for other antigen, human IgG1, 1133-type and 1131-typeof the humanized anti-Campath antibody Campath-1H were prepared tocompare their CDC activity.

(1) Construction of Expression Vector Comprising the Nucleotide SequenceEncoding 1133-Type Anti-Campath Domain-Swapped Antibody

An expression vector comprising the nucleotide sequence of a 1133-typeanti-Campath domain-swapped antibody which specifically recognizes humanCampath antigen (CD52), wherein among the amino acid sequences of theheavy chain constant region, CH1 and hinge are amino acid sequences ofhuman IgG1, and CH2 and CH3 are amino acid sequences of human IgG3, wasconstructed by the procedure shown below (FIG. 27).

Firstly, the amino acid sequences and the nucleotide sequences of theheavy chain variable region (Accession: 579311) and the light chainvariable region (Accession: S79307) of the humanized anti-Campathantibody Campath-1H were obtained from the data base of National Centerof Biotechnology Information (NCBI). The amino acid sequence of theheavy chain variable region of the humanized anti-Campath antibodyCampath-1H and the nucleotide sequence thereof are shown in SEQ IDNOs:58 and 59, respectively, and the amino acid sequence of the lightchain variable region of the humanized anti-Campath antibody Campath-1Hand the nucleotide sequence thereof are shown in SEQ ID NOs:60 and 61,respectively. Based on the sequence information, the amino acid sequenceof the heavy chain of the 1133-type anti-Campath domain-swapped antibodyrepresented by SEQ ID NO:62 containing sequences of the heavy chainvariable region of the humanized anti-Campath antibody Campath-1H andthe 1133-type heavy chain constant region, and the amino acid sequenceof the light chain of the anti-Campath antibody represented by SEQ IDNO:63 containing sequences of the light chain variable region of thehumanized anti-Campath antibody Campath-1H and the light chain constantregion of the humanized antibody were designed.

Next, the nucleotide sequence represented by SEQ ID NO:64 was designed.The sequence is a nucleotide sequence in which a restriction enzyme NotIrecognition sequence was added to the 5′-terminal side of the nucleotidesequence of the heavy chain variable region in the humanizedanti-Campath antibody Campath-1H, represented by SEQ ID NO:59, and arestriction enzyme ApaI recognition sequence to the 3′-terminal sidethereof. In addition, the nucleotide sequences represented by SEQ IDNOs:65, 66, 67 and 68 were designed based on the nucleotide sequencerepresented by SEQ ID NO:64. These sequences are nucleotide sequencesdesigned by dividing the nucleotide sequence represented by SEQ ID NO:64into four parts, in such a manner that mutually adjoining sequences havean overlapping sequence of approximately 20 bp and the sense chain andantisense chain are reciprocated.

In fact, each of synthetic oligo DNAs of the nucleotide sequencesrepresented by SEQ ID NOs:65, 66, 67 and 68 was prepared (manufacturedby FASMAC), and PCR was carried out using them. By preparing a reactionsolution for PCR [0.02 unit/μl KOD+DNA Polymerase (manufactured byTOYOBO), 0.2 mM dNTPs, 1 mM magnesium chloride, 1/10 volume of 10-foldconcentrated PCR Buffer (manufactured by TOYOBO, attached to the KOD DNAPolymerase)] in such a manner that the two synthetic oligo DNAspositioned at both terminals respectively became a final concentrationof 0.5 μM and the other two synthetic oligo DNAs positioned insidethereof respectively became a final concentration of 0.1 μM, and PCR wascarried out using a DNA thermal cycler GeneAmp PCR System 9700(manufactured by Applied Biosystems) by heating at 94° C. for 4 minutes,followed by 25 cycles of 3 steps of reactions at 94° C. for 30 seconds,at 50° C. for 30 seconds and at 68° C. for 60 seconds. After completionof the PCR, the reaction solution was subjected to agarose gelelectrophoresis, and a PCR product of about 480 bp was recovered usingQIAquick™ Gel Extraction Kit (manufactured by QIAGEN). The thusrecovered PCR product was digested with restriction enzymes Nod(manufactured by Takara Shuzo) and ApaI (manufactured by Takara Shuzo),and then the reaction solution was subjected to agarose gelelectrophoresis, and a DNA fragment of about 450 bp was cleaved andpurified using QIAquick™ Gel Extraction Kit (manufactured by QIAGEN). Onthe other hand, a DNA fragment of about 13 kbp was cleaved and purifiedby carrying out the same restriction enzyme treatment on the expressionvector plasmid of 1133-type anti-CD20 domain-swapped antibody preparedin the item 2 of Example 2. After mixing these purified DNA fragments, aligation reaction was carried out by adding Ligation High solution(manufactured by TOYOBO), and Escherichia coli XL1-BLUE MRF′(manufactured by Stratagene) was transformed using the reactionsolution. Each plasmid DNA was prepared from the thus obtainedtransformant clones and allowed to react using Big Dye Terminator Cycle™Sequencing Kit v3.1 (manufactured by Applied Biosystems) in accordancewith the instructions attached thereto, and then the nucleotide sequenceof the DNA inserted into each plasmid was analyzed by a DNA sequencerABI PRISM 3700™ DNA Analyzer of the same company to confirm that a1133-type expression vector plasmid in which the heavy chain variableregion was replaced by a nucleotide sequence encoding the heavy chainvariable region of the humanized anti-Campath antibody Campath-1H wasobtained.

Next, the nucleotide sequence represented by SEQ ID NO:69 was designed.The sequence is a nucleotide sequence in which a recognition sequencerestricted by a restriction enzyme EcoRI was added to the 5′-terminalregion of the nucleotide sequence of the light chain variable region inthe humanized anti-Campath antibody Campath-1H represented by SEQ IDNO:61, and a recognition sequence restricted by a restriction enzymeBsiWI to the 3′-terminal region thereof. In addition, each of thenucleotide sequences represented by SEQ ID NOs:70, 71, 72 and 73 wasdesigned based on the nucleotide sequence represented by SEQ ID NO:69.These sequences were nucleotide sequences designed by dividing thenucleotide sequence represented by SEQ ID NO:69 into four parts, in sucha manner that mutually adjoining sequences have an overlapping sequenceof approximately 20 bps and the sense chain and antisense chain werereciprocated. By carrying out PCR using four synthetic oligo DNAfragments represented by these nucleotide sequences, they were ligatedvia the overlapping sequence of mutually adjoining sequences to amplifya DNA fragment having the nucleotide sequence represented by SEQ IDNO:69.

In fact, each of synthetic oligo DNA fragments of the nucleotidesequences represented by SEQ ID NOs:70, 71, 72 and 73 were prepared(manufactured by FASMAC), and PCR was carried out using them. Bypreparing a reaction solution for PCR [0.02 unit/μl KOD+DNA Polymerase(manufactured by TOYOBO), 0.2 mM dNTPs, 1 mM magnesium chloride, 1/10volume of 10-fold concentrated PCR Buffer (manufactured by TOYOBO,attached to the KOD DNA Polymerase)] in such a manner that the twosynthetic oligo DNAs positioned at both terminals respectively became afinal concentration of 0.5 μM and the other two synthetic oligo DNAspositioned inside thereof respectively became a final concentration of0.1 μM, and PCR was carried out using a DNA thermal cycler GeneAmp PCRSystem 9700™ (manufactured by Applied Biosystems) by heating at 94° C.for 4 minutes, followed by 25 cycles of 3 steps of reactions at 94° C.for 30 seconds, at 50° C. for 30 seconds and at 68° C. for 60 seconds.After completion of the PCR reaction, the reaction solution wassubjected to agarose gel electrophoresis, and a PCR product of about 420bp was recovered using QIAquick™ Gel Extraction Kit (manufactured byQIAGEN). The thus recovered PCR product was digested with restrictionenzymes EcoRI (manufactured by Takara Shuzo) and BsiWI (manufactured byTOYOBO), and then the reaction solution was subjected to agarose gelelectrophoresis, and a DNA fragment of about 400 bp was cleaved andpurified using QIAquick™ Gel Extraction Kit (manufactured by QIAGEN). Onthe other hand, a DNA fragment of about 13 kbp was cleaved and purifiedby carrying out the same restriction enzyme treatment on the 1133-typeexpression vector plasmid in which the heavy chain variable region wasreplaced by a nucleotide sequence encoding the heavy chain variableregion of the humanized anti-Campath antibody Campath-1H, prepared inthis item. After mixing these purified DNA fragments, a ligationreaction was carried out by adding Ligation High solution (manufacturedby TOYOBO), and Escherichia coli XL1-BLUE MRF′ (manufactured byStratagene) was transformed using the reaction solution. Each plasmidDNA was prepared from the thus obtained transformant clones and allowedto react using Big Dye Terminator Cycle™ Sequencing Kit v3.1(manufactured by Applied Biosystems) in accordance with the instructionsattached thereto, and then the nucleotide sequence of the DNA insertedinto each plasmid was analyzed by a DNA sequencer ABI PRISM 3700™ DNAAnalyzer of the same company to confirm that expression vector plasmidfor 1133-type anti-Campath antibody, pKTX93/Campath1H-1133 was obtained.

(2) Construction of Expression Vector Comprising the Nucleotide SequenceEncoding Human IgG Anti-Campath Antibody

An expression vector comprising the nucleotide sequence of ananti-Campath human IgG1 antibody which specifically recognizes a humanCampath antigen (CD52), wherein the heavy chain constant region was theamino acid sequence of human IgG1, was constructed by the procedureshown bellow (FIG. 28).

The expression vector plasmid for 1133-type anti-Campath antibody,pKTX93/Campath1H-1133 prepared in this item was digested withrestriction enzymes EcoRI (manufactured by Takara Shuzo) and ApaI(manufactured by Takara Shuzo), and then the reaction solution wassubjected to agarose gel electrophoresis, and a DNA fragment of about3,300 bp was cleaved and purified using QIAquick™ Gel Extraction Kit(manufactured by QIAGEN). On the other hand, a DNA fragment of about 10kbp was cleaved and purified by carrying out the same restriction enzymetreatment on the expression vector plasmid for anti-CD20 human IgG1chimeric antibody, pKANTEX2B8P. After mixing these purified DNAfragments, a ligation reaction was carried out by adding Ligation Highsolution (manufactured by TOYOBO), and Escherichia coli XL1-BLUE MRF′(manufactured by Stratagene) was transformed using the reactionsolution. Each plasmid DNA was prepared from the thus obtainedtransformant clones and allowed to react using Big Dye Terminator Cycle™Sequencing Kit v3.1 (manufactured by Applied Biosystems) in accordancewith the instructions attached thereto, and then the nucleotide sequenceof the DNA inserted into each plasmid was analyzed by a DNA sequencerABI PRISM 3700™ DNA Analyzer of the same company to confirm thatexpression vector plasmid for anti-Campath human IgG1 antibody,pKTX93/Campath1H-IgG1 was obtained.

(3) Construction of Expression Vector Comprising the Nucleotide SequenceEncoding 1133-Type Anti-Campath Antibody

An expression vector comprising the nucleotide sequence of a human1133-type anti-Campath antibody which specifically recognizes a humanCampath antigen (CD52), wherein among the amino acid sequences of theheavy chain constant region, CH1 and hinge are the amino acid sequencesof human IgG1, CH2 is the amino acid sequence of human IgG3 and CH3 isthe amino acid sequence of human IgG1, was constructed by the procedureshown bellow (FIG. 29).

The expression vector plasmid for 1133-type anti-Campath antibody,pKTX93/Campath1H-1133 prepared in this item was digested withrestriction enzymes EcoRI (manufactured by Takara Shuzo) and ApaI(manufactured by Takara Shuzo), and then the reaction solution wassubjected to agarose gel electrophoresis, and a DNA fragment of about3,300 bp was cleaved and purified using QIAquick™ Gel Extraction Kit(manufactured by QIAGEN). On the other hand, a DNA fragment of about 10kbp was cleaved and purified by carrying out the same restriction enzymetreatment on the expression vector plasmid for 1131-type anti-CD20antibody, pKTX93/1131 prepared in the item 1 of Example 3. After mixingthese purified DNA fragments, a ligation reaction was carried out byadding Ligation High solution (manufactured by TOYOBO), and theEscherichia coli XL1-BLUE MRF′ (manufactured by Stratagene) wastransformed using the reaction solution. Each plasmid DNA was preparedfrom the thus obtained transformant clones and allowed to react usingBig Dye Terminator Cycle™ Sequencing Kit v3.1 (manufactured by AppliedBiosystems) in accordance with the instructions attached thereto, andthen the nucleotide sequence of the DNA inserted into each plasmid wasanalyzed by a DNA sequencer ABI PRISM 3700™ DNA Analyzer of the samecompany to confirm that expression vector plasmid for 1131-typeanti-Campath domain-swapped antibody, pKTX93/Campath1H-1131 wasobtained.

2. Stable Expression of Anti-Campath Human IgG1 Antibody, 1133-TypeAnti-Campath Domain-Swapped Antibody and 1131-Type Anti-CampathDomain-Swapped Antibody in Animal Cell

Each of the expression vectors for the anti-Campath human IgG1 antibody,the 1133-type anti-Campath domain-swapped antibody and the 1131-typeanti-Campath domain-swapped antibody prepared in the item 1 of thisExample was introduced into the host cell CHO/FUT8^(−/−) described inthe item 3 of Example 1, and a cell capable of stably producing theanti-Campath human IgG1 antibody, the 1133-type anti-Campathdomain-swapped antibody or the 1131-type anti-Campath domain-swappedantibody was prepared in the same manner as in the item 3 of Example 1.

3. Purification of Anti-Campath Human IgG1 Antibody, 1133-TypeAnti-Campath Domain-Swapped Antibody and 1131-Type Anti-CampathDomain-Swapped Antibody

Each of the transformants capable of expressing the anti-Campath humanIgG1 antibody, the 1133-type anti-Campath domain-swapped antibody or the1131-type anti-Campath domain-swapped antibody, obtained in the item 2of this Example, was cultured and purified in the same manner as in theitem 5 of Example 1. Corresponding expression vectors, host cells andnames of the purified antibodies of each of the modified antibodies areshown in Table 9.

TABLE 9 Expression vector Host cell Purified antibody (name)pKTX93/Campath1H-IgG1 Ms705 Campath1H-IgG1 pKTX93/Campath1H-1133 Ms705Campath1H-1133 pKTX93/Campath1H-1131 Ms705 Campath1H-1131

4. Evaluation of the Purification Degree of Various Anti-CampathAntibodies by SDS-PAGE

In order to evaluate purification degree of the purified samples of thevarious modified antibodies obtained in the item 3 of this Example,SDS-PAGE was carried out in the same manner as in the item 6 of Example1 to thereby confirm that the desired IgG molecule constituted by therespective H chain and L chain was contained at a sufficient ratio ineach of the purified modified antibody samples obtained in the item 3 ofthis Example.

Example 10 Measurement of the CDC Activity of Anti-Campath Human IgG1Antibody, 1133-Type Anti-Campath Domain-Swapped Antibody and 1131-TypeAnti-Campath Domain-Swapped Antibody:

Using the purified samples of the various anti-Campath antibodiesCampath1H-IgG1, Campath1H-1133 and Campath1H-1131 obtained in the item 3of Example 9, their in vitro CDC activity to the Campathantigen-positive CLL cell lines MEC-1, MEC-2 and EHEB was measured. Whenthe test was carried out in the same manner as in Example 8,Campath1H-1133 and Campath1H-1131 showed higher CDC activity than thatof Campath1H-IgG for all of the cell lines MEC-1, MEC-2 and EHEB.

The present invention provides a recombinant antibody composition havinghigher complement-dependent cytotoxic activity than a human IgG1antibody and a human IgG3 antibody, wherein a polypeptide comprising aCH2 domain in the Fc region of a human IgG1 antibody is replaced by apolypeptide comprising an amino acid sequence which corresponds to thesame position of a human IgG3 antibody indicated by the EU index as inKabat, et al., a DNA encoding an antibody molecule contained in therecombinant composition or a heavy chain constant region of the antibodymolecule; a transformant obtainable by introducing the DNA into a hostcell; a process for producing the recombinant antibody composition usingthe transformant; and a medicament comprising the recombinant antibodycomposition as an active ingredient

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skill in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

1. A recombinant antibody composition, comprising recombinant antibodieshaving higher complement-dependent cytotoxic activity than human IgG1and human IgG3 antibodies, when determined under same conditions, saidhuman IgG1 and human IgG3 antibodies having a same variable regionbinding to an antigen as said recombinant antibodies, wherein saidrecombinant antibodies are a variant of the human IgG1 antibodycomprising domains of CH1, hinge, CH2, and CH3, said domains of CH1,hinge, CH2, and CH3 consisting of the amino acids of SEQ ID NO: 76 asshown in FIG. 1, wherein at least first 111 amino acid residues fromN-terminal of amino acids at positions 240-447 of SEQ ID NO: 76 arereplaced with a same number of amino acid residues at correspondingpositions of SEQ ID NO: 78 as shown in FIG.
 1. 2. The recombinantantibody composition according to claim 1, wherein said recombinantantibodies have complex type N-glycoside-linked sugar chains in the Fcregion, wherein the ratio of sugar chains in which fucose is not boundto N-acetylglucosamine in the reducing terminal of the sugar chainsamong the total complex type N-glycoside-linked sugar chains which bindto the Fc region contained in the composition is 20% or more.
 3. Therecombinant antibody composition according to claim 1, wherein saidrecombinant antibodies have complex type N-glycoside-linked sugar chainsin the Fc region, wherein the complex type N-glycoside-linked sugarchains bound to the Fc region of the antibody are sugar chains in whichfucose is not bound to N-acetylglucosamine in the reducing terminal inthe sugar chains.
 4. A medicament comprising the recombinant antibodycomposition described in claim 1 as an active ingredient.